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Regulating the plant innate immune system : the roles of three Arabidopsis MUSE proteins Johnson, Kaeli 2016

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REGULATING THE PLANT INNATE IMMUNE SYSTEM:  THE ROLES OF THREE ARABIDOPSIS MUSE PROTEINS  by  Kaeli Johnson  B.Sc. (Honours), Queen’s University, 2010  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Botany)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2016  © Kaeli Johnson, 2016 ii  Abstract  The plant innate immune system is highly effective in impeding infection by a broad spectrum of microbial pathogens. Strict regulation of immune signaling in plants is required to both facilitate rapid defense response induction upon pathogen detection and prevent the precocious activation of immunity, the latter of which has associated fitness costs. Despite their significance, the regulatory mechanisms governing plant immunity have only been partially characterized. Previously, members of the Li research group employed a forward genetic screen to identify positive regulators of innate immunity. This suppressor screen was performed using the unique Arabidopsis autoimmune mutant snc1 (suppressor of npr1, constitutive 1), which contains a gain-of-function mutation in an NLR (NOD-LIKE RECEPTOR) protein. The identified MOS (MODIFIER OF SNC1) genes highlighted the importance of diverse biological processes in the regulation of disease resistance. More recently, a snc1 enhancer screen was conducted to identify negative regulators of plant immune signaling. This thesis describes the cloning and characterization of three mutants isolated from this MUSE (MUTANT, SNC1-ENHANCING) screen. The muse9 mutant carries a molecular lesion in the gene encoding the chromatin remodeler SPLAYED (SYD). Molecular analyses showed that SYD negatively regulates SNC1 expression and thus functions antagonistically to MOS1 and MOS9, both of which were previously shown to positively regulate SNC1 transcription. This study emphasizes the importance of finely-tuned transcriptional control in NLR-mediated immunity.   The muse4 mutant contains a partial loss-of-function mutation in NRPC7, which encodes an RNA polymerase III (Pol III) subunit. This is the first reported viable Pol III mutant. Using iii  RT-PCR, it was established that the mutation in NRPC7 affects the expression of a diverse suite of genes and results in distortions in alternative splicing.  The mutation responsible for the muse7 phenotypes is in a gene that encodes a protein of unknown function. MUSE7 negatively regulates SNC1 at the protein level, although no interactions were detected between MUSE7 and other known regulators of NLR protein turnover. This suggests that MUSE7 either regulates protein synthesis or is involved in an alternate degradation pathway.  Taken together, these characterizations underscore the complexity inherent in the molecular mechanisms that control plant immune signaling.       iv  Preface  The research comprising this thesis is the result of work performed between September 2010 and April 2016. Section 1.5 of Chapter 1 as well as Chapters 2 and 3 have been previously published and a manuscript corresponding to Chapter 4 is currently in preparation for publication. The details of these manuscripts and the contributions of the candidate are as follows:  Johnson, K.C.M., Dong, O.X., Huang, Y., and Li, X. (2012) A rolling stone gathers no moss, but resistant plants must gather their MOSes. Cold Spring Harbor Symposia on Quantitative Biology 77:259-68.  The candidate wrote and edited the majority of the manuscript. O.X. Dong wrote Section 1.5.5 and created Figure 1.1. Y. Huang contributed to Sections 1.5.1 and wrote Sections 1.5.2 and 1.5.7. X. Li supervised the preparation of the manuscript.  Johnson, K.C.M.*, Xia, S.*, Feng, X., and Li, X. (2015) The chromatin remodeler SPLAYED negatively regulates SNC1-mediated immunity. Plant and Cell Physiology 56:1616-23 (*Co-first authorship).  The candidate performed most of the experiments and wrote the manuscript. S. Xia performed the positional cloning of the muse9 mutant. X. Feng (JIC, Norwich) conducted DNA methylation analyses. X. Li (UBC, Vancouver) supervised the work performed by S. Xia and the candidate, as well as the preparation of the manuscript.  v  Johnson, K.C.M., Yu, Y., Gao, L., Eng, R.C., Wasteneys, G.O., Chen, X., and Li, X. (2016) A partial loss-of-function mutation in an RNA polymerase III subunit leads to pleiotropic defects. Journal of Experimental Botany 67(8):2219-30.  The candidate performed most of the experiments and wrote the manuscript. Y. Yu performed small RNA library preparations and northern blotting. L. Gao conducted small RNA library analyses. R.C. Eng performed confocal microscopy using transgenic lines generated by the candidate. X. Chen (UCR, Riverside) supervised work performed by Y. Yu, L. Gao, and the candidate. G.O. Wasteneys (UBC, Vancouver) supervised work performed by R.C. Eng. X. Li (UBC, Vancouver) supervised work performed by the candidate and the preparation of the manuscript.   Johnson, K.C.M., Zhao, J., Roth, C., Wiermer, M., and Li, X. The putative casein kinase II substrate MUSE7 negatively regulates the accumulation of SNC1. Manuscript in preparation.  The candidate performed most of the experiments and wrote the manuscript. J. Zhao performed the positional cloning of the muse7 mutant. C. Roth conducted confocal microscopy using transgenic lines generated by the candidate. M. Wiermer (GAU, Göttingen) supervised the work performed by C. Roth. X. Li (UBC, Vancouver) supervised the work performed by J. Zhao and the candidate, as well as the preparation of the manuscript.     vi  Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ......................................................................................................................... vi List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Abbreviations ................................................................................................................. xiv Acknowledgements ................................................................................................................... xvii Dedication ................................................................................................................................... xix Chapter 1: An introduction to plant innate immunity ...............................................................1 1.1 Significance..................................................................................................................... 1 1.2 Physical and chemical barriers........................................................................................ 2 1.3 Immunity triggered by pathogen-associated molecular patterns .................................... 4 1.4 Immunity triggered by effector recognition .................................................................... 6 1.4.1 Overview ..................................................................................................................... 6 1.4.2 The structure of NOD-like receptor (NLR) proteins .................................................. 6 1.4.3 Effector detection ........................................................................................................ 7 1.4.4 NLR protein activation ............................................................................................... 9 1.4.5 Downstream signaling .............................................................................................. 10 1.5 Positive regulation of SNC1-mediated immunity ......................................................... 11 1.5.1 Overview ................................................................................................................... 11 1.5.2 Regulators of SNC1 gene expression levels: MOS1 and MOS9 .............................. 14 vii  1.5.3 Components of the RNA processing machinery: MOS2, MOS4, and MOS12 ........ 15 1.5.4 Nuclear proteins important for mRNA export: The Nup107-160 complex and MOS11 .................................................................................................................................. 19 1.5.5 Components involved in nucleocytoplasmic protein trafficking: MOS6, MOS7, and MOS14 .................................................................................................................................. 21 1.5.6 Transcriptional co-repression with SNC1: MOS10 (TPR1) ..................................... 23 1.5.7 Protein modifying enzymes: MOS5 and MOS8 ....................................................... 24 1.5.8 Integration of the MOS genes ................................................................................... 26 1.6 Thesis objectives ........................................................................................................... 29 Chapter 2: The chromatin remodeler SPLAYED negatively regulates SNC1-mediated immunity .......................................................................................................................................30 2.1 Summary ....................................................................................................................... 30 2.2 Introduction ................................................................................................................... 31 2.3 Results ........................................................................................................................... 34 2.3.1 Isolation of muse9 snc1 mos4 ................................................................................... 34 2.3.2 Phenotypes associated with muse9 result from a point mutation in SYD ................. 35 2.3.3 The syd-4 single mutant displays enhanced disease resistance ................................ 39 2.3.4 Mutations in SYD result in elevated transcription of SNC1 ...................................... 42 2.4 Discussion ..................................................................................................................... 43 2.5 Materials and methods .................................................................................................. 49 2.5.1 Plant growth conditions and mutant isolation ........................................................... 49 2.5.2 Expression analysis ................................................................................................... 49 2.5.3 Positional cloning...................................................................................................... 50 viii  2.5.4 Pathogen assays ........................................................................................................ 50 2.5.5 Genetic crosses.......................................................................................................... 51 Chapter 3: A partial loss-of-function mutation in an Arabidopsis RNA polymerase III subunit leads to pleiotropic defects ............................................................................................52 3.1 Summary ....................................................................................................................... 52 3.2 Introduction ................................................................................................................... 53 3.3 Results ........................................................................................................................... 56 3.3.1 The isolation, characterization, and identification of the muse4/nrpc7-1 mutant ..... 56 3.3.2 The mutation at an intron/exon junction of NRPC7 results in intron retention and is responsible for the muse4 phenotypes .................................................................................. 59 3.3.3 Splicing of SNC1 is altered in the nrpc7-1 mos4 snc1 background .......................... 63 3.3.4 The nrpc7-1 single mutant does not have altered immune responses ...................... 66 3.3.5 nrpc7-1 has global defects in RNA levels ................................................................ 67 3.3.6 NRPC7 localizes to the nucleus ................................................................................ 71 3.3.7 nrpc7-1 has pleiotropic developmental defects ........................................................ 72 3.4 Discussion ..................................................................................................................... 74 3.5 Materials and methods .................................................................................................. 78 3.5.1 Plant growth conditions and mutant isolation ........................................................... 78 3.5.2 Total RNA extraction and analysis ........................................................................... 78 3.5.3 Infection assays ......................................................................................................... 79 3.5.4 Positional cloning and Illumina whole-genome sequencing .................................... 79 3.5.5 Preparation of transgenic plants and confocal microscopy ....................................... 80 3.5.6 Yeast complementation ............................................................................................. 80 ix  3.5.7 Small RNA library construction and sequencing...................................................... 81 3.5.8 Analysis of small RNA high throughput sequencing data ........................................ 81 Chapter 4: The putative kinase substrate MUSE7 negatively regulates the accumulation of SNC1..............................................................................................................................................83 4.1 Summary ....................................................................................................................... 83 4.2 Introduction ................................................................................................................... 84 4.3 Results ........................................................................................................................... 87 4.3.1 The muse7 mutation re-establishes snc1-like phenotypes in the mos4 snc1 background ............................................................................................................................ 87 4.3.2 MUSE7 encodes an uncharacterized protein conserved amongst eukaryotes .......... 88 4.3.3 Two independent muse7 single mutant lines exhibit enhanced disease resistance ... 93 4.3.4 MUSE7 localizes to both the nucleus and the cytoplasm ......................................... 93 4.3.5 Mutations in MUSE7 affect SNC1 accumulation ..................................................... 96 4.3.6 MUSE7 does not appear to interact with known regulators of SNC1 turnover ........ 98 4.4 Discussion ..................................................................................................................... 99 4.5 Methods and materials ................................................................................................ 102 4.5.1 Plant growth conditions and mutant isolation ......................................................... 102 4.5.2 Positional cloning.................................................................................................... 102 4.5.3 Total RNA extraction and analysis ......................................................................... 103 4.5.4 Infection assays ....................................................................................................... 103 4.5.5 Preparation of transgene constructs and plant transformation ................................ 104 4.5.6 Protein extraction and co-immunoprecipitation...................................................... 104 Chapter 5: Final perspectives ...................................................................................................106 x  5.1 Overview ..................................................................................................................... 106 5.2 Immunoregulatory mechanisms examined in this thesis ............................................ 109 5.2.1 Chromatin architecture and transcriptional modulation ......................................... 109 5.2.1.1 Findings from the MUSE9/SPLAYED study ................................................. 109 5.2.1.2 Future directions ............................................................................................. 110 5.2.2 Alternative splicing of genes encoding NLR proteins ............................................ 111 5.2.2.1 Findings from the MUSE4/NRPC7 study ...................................................... 111 5.2.2.2 Future directions ............................................................................................. 112 5.2.3 NLR protein accumulation ...................................................................................... 112 5.2.3.1 Findings from the MUSE7 study .................................................................... 112 5.2.3.2 Future directions ............................................................................................. 114 5.3 Summary ..................................................................................................................... 115 References ...................................................................................................................................116  xi  List of Tables  Table 4.1 MUSE7 homologs are present in low copy number across land plants. ....................... 91  xii  List of Figures  Figure 1.1 A model depicting the involvement of the MOS proteins in plant immunity ............. 26   Figure 2.1 Phenotypic analysis of the muse9 mos4 snc1 triple mutant ........................................ 35 Figure 2.2 Positional cloning of the MUSE9 locus on chromosome 2 ......................................... 37 Figure 2.3 MUSE9 encodes SYD, an ATP-dependent chromatin remodeller .............................. 38 Figure 2.4 The syd-10 single mutant does not display enhanced disease resistance .................... 41 Figure 2.5 Alignment of SYD proteins from a number of plant species ...................................... 42 Figure 2.6 SNC1 protein levels in the indicated genotypes .......................................................... 43 Figure 2.7 CHH methylation in wild type, syd-4, rdr2, and ddm1 plants near the SNC1 locus ... 47 Figure 2.8 SYD functions antagonistically with MOS1/MOS9 to regulate SNC1 transcription .. 48 Figure 3.1 Characterization of the muse4 mos4 snc1 triple mutant .............................................. 57 Figure 3.2 Map-based cloning of the muse4 locus on chromosome 1 .......................................... 59 Figure 3.3 MUSE4 is NRPC7........................................................................................................ 60 Figure 3.4 Sequence alignment of RPC25 from a broad range of species ................................... 61 Figure 3.5 Yeast complementation with NRPC7 .......................................................................... 62 Figure 3.6 Splicing defects in nrpc7-1 .......................................................................................... 64 Figure 3.7 SNC1 gene and protein expressionin nrpc7-1 ............................................................. 65 Figure 3.8 Immune characterization of nrpc7-1 single mutant plants .......................................... 67 Figure 3.9 Global RNA defects in nrpc7-1 ................................................................................... 68 Figure 3.10 RNA defects in nrpc7-1 ............................................................................................. 69 Figure 3.11 Subcellular localization of NRPC7-GFP ................................................................... 72 Figure 3.12 Developmental defects of the nrpc7-1 mutant .......................................................... 73 xiii  Figure 4.1 Phenotypic characterization of muse7 mos4 snc1 ....................................................... 88 Figure 4.2 Positional cloning of MUSE7 ...................................................................................... 90 Figure 4.3 Multiple alignment of MUSE7 homolog amino acid sequences ................................. 92 Figure 4.4 Characterization of two independent muse7 single mutant alleles .............................. 94 Figure 4.5 Characterization of muse7 developmental phenotypes ............................................... 95 Figure 4.6 Subcellular localization of MUSE7-GFP .................................................................... 96 Figure 4.7 Regulation of SNC1 by MUSE7 ................................................................................. 97 Figure 4.8 MUSE7 does not co-immunoprecipitate with HSP90.3, CPR1, or SNC1 .................. 99 Figure 5.1 A model depicting the involvement of MOSes and MUSEs in plant immunity ....... 108       xiv  List of Abbreviations  ABA  abscisic acid ABRC  Arabidopsis biological resource center ADR  activated disease resistance ATXR7 Arabidopsis trithorax-related 7 Avr  avirulence AvrB  avirulence gene from Pseudomonas syringae pv. glycinea AvrPphB avirulence gene from Pseudomonas syringae pv. phaseolicola AvrRpm1 avirulence gene from Pseudomonas syringae pv. syringae AvrRps4 avirulence gene from Pseudomonas syringae pv. pisi AvrRpt2 avirulence gene from Pseudomonas syringae pv. tomato BAK1  BRI1-associated receptor kinase 1 BAT2  HLA-B associated transcript 2 bHLH84 basic helix-loop-helix 84 BIK1  Botrytis-induced kinase 1 BRM  brahma CC  coiled-coil cDNA  complementary DNA CERK1 chitin elicitor receptor kinase 1 CESA3 cellulose synthase 3 CHS3  chilling sensitive 3 CNL  CC-NB-LRR Col-0  Columbia ecotype of Arabidopsis thaliana CPR  constitutive expresser of PR genes CSA1  constitutive shade-avoidance 1 CUC  cup-shaped cotyledons DAMP  damage-associated molecular pattern DDM1  decrease in DNA methylation 1 DNA  deoxyribonucleic acid DND  defense no death EDS1  enhanced disease susceptibility 1 EMS  ethyl methanesulfonate ERA1  enhanced response to abscisic acid 1 ET  ethylene ETI  effector-triggered immunity FLAG  epitope tag with the amino acid sequence DYKDDDDK FLC  flowering locus C FLS2  flagellin-sensitive 2 GFP  green fluorescence protein GUS  β-glucuronidase H.a.  Hyaloperonospora arabidopsidis HA  hemagglutinin xv  HDA19 histone deacetylase 19 HD-ZIP homeodomain leucine zipper HopA1 avirulence gene from Pseudomonas syringae pv. syringae HopF2  avirulence gene from Pseudomonas syringae pv. tomato HR  hypersensitive response HSP  heat shock protein JA  jasmonate LAZ5  lazarus 5 Ler  Landsberg erecta ecotype of Arabidopsis thaliana LIM  “Lin11, Isl-1, Mec3” domain LRR  leucine-rich repeat MAC  MOS4-associated complex MAMP microbe-associated molecular pattern miRNA microRNA MLA10 mildew A 10 MOS  modifier of snc1 mRNA  messenger RNA MS  Murashige and Skoog MUSE  mutant, snc1-enhancer MYC  epitope tag derived from the human myelocytomatosis viral oncogene N  Nicotiana (N protein in tobacco) NB  nucleotide-binding NDR1  non race-specific disease resistance 1 NLR  NOD-like receptor NLS  nuclear localization signal NOD  nucleotide-binding oligomerization domain NPC  nuclear pore complex NPR1  non-expresser of PR genes 1 NRPC7 nuclear RNA polymerase C subunit 7 Nup  nucleoporin P.s.m.  Pseudomonas syringae pv. maculicola P.s.t.  Pseudomonas syringae pv. tomato PAD4  phytoalexin-deficient 4 PAGE  polyacrylamide gel electrophoresis PAMP  pathogen-associated molecular pattern PBS1  AvrPphB susceptible 1 PDAP1 PDGF-associated protein 1 PDGF  platelet-derived growth factor PEPR  PEP1 receptor PHB  phabulosa Pol  RNA polymerase PopP2  avirulence gene from Ralstonia solanacearum PR  pathogenesis-related PRP  pre-mRNA processing PRR  pattern recognition receptor xvi  PTI  PAMP-triggered immunity PTM  post-translational modification qPCR  quantitative polymerase chain reaction R  resistance REV  revulota RGA  resistance gene analog RIN4  RPM1-interacting protein 4 RLK  receptor-like kinase RLP  receptor-like protein RNA  ribonucleic acid ROS  reactive oxygen species Rpc  RNA polymerase core protein RPL18  ribosomal protein L18 RPM1  resistance to Pseudomonas syringae pv. maculicola RPP  recognition of Peronospora parasitica RPS  resistant to Pseudomonas syringae rRNA  ribosomal RNA RRS1  resistant to Ralstonia solanacearum 1 SA  salicylic acid SAG101 senescence-associated gene 101 SAR  systemic acquired resistance SCF  SKP1-CULLIN-F-box complex SDG8  set domain group 8 SDS  sodium dodecyl sulphate siRNA  small interfering RNA SNC1  suppressor of npr1, constitutive 1 snRNA small nuclear RNA SPL6  squamosa promoter binding protein-domain transcription factor 6 SPP2  spliceosomal protein 2 SR  serine-arginine rich protein STAND signal transduction ATPases with numerous domains SWI/SNF switch/sucrose non-fermentable SWR1  SWI2/SNF2 related 1 complex SYD  splayed T-DNA transfer-DNA TAIR  the Arabidopsis information resource TIR  Toll Interleukin receptor TNL  TIR-NB-LRR TPL  topless TPR1  topless-related 1 TRN  transportin tRNA  transfer RNA UBQ  ubiquitin WRKY “WRKY” domain Ws  Wassilewskija ecotype of Arabidopsis thaliana xvii  Acknowledgements  The completion of this thesis was only possible with the assistance of many individuals.  Foremost, I would like to express my gratitude to my supervisor Dr. Xin Li for her expertise, patience, and enduring support. Her excited approach to research is infectious, and her mix of intelligence, kindness, and humour make her a uniquely gifted mentor. I consider myself lucky to have spent these past six years learning from her.  I would also like to thank my committee members Dr. Carl Douglas, Dr. Brian Ellis, and Dr. Ljerka Kunst for their advice and encouragement. Each of my research projects benefitted substantially from their insightful questions and thoughtful suggestions. I also want to thank Dr. Xuemei Chen, who kindly hosted me at the University of California, Riverside. Dr. Yuelin Zhang served as another invaluable resource. While at the National Institute of Biological Sciences in Beijing, P.R. China, his laboratory performed next-generation sequencing and analysis which was instrumental in advancing the research presented in this dissertation, and his counsel over the years has been much appreciated. I am also grateful to all members of the Li and Zhang labs, past and present, for their assistance and camaraderie. Express thanks are extended to Dr. Yu Ti Cheng, Dr. Fang Xu, and Dr. Jin Zhao for their mentorship in the early days of my degree, and to Patrick Gannon for initiating the MUSE genetic screen. Oliver Dong also deserves a special mention; my graduate school experience would not have been the same without the constant company and support of my “lab brother”. I would like to offer my thanks to all members of the Michael Smith Laboratories and Department of Botany communities for making my experience at UBC both fruitful and enjoyable. xviii  Funding for this research was provided by NSERC Canada Graduate Scholarships (both Master’s and Doctoral), a UBC Four-Year Fellowship, a UBC Killam Doctoral Scholarship, and a Frances Chave Memorial Fellowship from the Department of Botany at UBC. I am grateful for all of the opportunities these funding sources have provided. The process of completing a doctorate would have been much more arduous and considerably less fun without the love and support of a number of friends. Erin Fenneman, Kate McGrath, Sara Miller, Jessica Lu, and Virginia Woloshen provided excellent dinner company and served as a sounding board for all of life’s most difficult problems. Dr. Ryan Eng, Caitlin Donnelly, Dr. Brandon Le, Dr. Maryam Sanei, and Zeina Waheed were always up for much-needed coffee breaks. Emily Brockman, Ellisha Cunningham, Alexander Edgar, Dr. Kyle Glenn, Courtney Holden, and Ilona Houston helped me maintain perspective and enjoy life away from the research bench.  I am lucky to have a large and inspiring family. I would like to thank Mom and Chickie, Dad and Jeannie, my grandparents, and my siblings (Angus, Heather, Jesse, Ian, Eric, and Tye) along with their respective partners. They have all been incredibly understanding during the course of this degree, and have offered nothing but encouragement and love even while navigating the ups and downs of their own lives.  And finally, thanks to my partner Mike Scott – words do not suffice.      xix  Dedication  To my mother, Robin Minion. 1  Chapter 1: An introduction to plant innate immunity1  1.1 Significance   Plants associate with numerous microbes in their surrounding environment, the vast majority of which do not elicit symptoms of disease. This is because plants possess a layered innate immune system that is tightly regulated, enabling appropriate and specific responses to most pathogenic threats without spurious immune activation that can result in fitness costs. However, the relationship between plants and potentially pathogenic microbes is a dynamic one, with both sides evolving rapidly in an effort to detect and avoid detection, respectively. The ability of plants to defend themselves against infection has a direct societal impact as plant diseases cause significant reductions in crop yields; estimates vary, but global food production is thought to incur a loss of approximately 10% annually due to plant pathogens (Scott & Strange 2005). In addition to potentially limiting food availability, crop losses can destabilize the economy in regions that are highly dependent on food production as a source of employment. Climate change is likely to exacerbate this issue, as studies have demonstrated that predicted changes in carbon dioxide levels, temperature, and drought conditions may increase both disease prevalence and severity (reviewed in Gregory et al. 2009). Current agricultural practices involve the use of pesticides and fungicides to constrain pathogen proliferation. However, concerns have been raised over the long-term, intensive use of agrichemicals based on potential risks to human health and the environment. Additionally, over time these strategies may lead to the development                                                  1 A version of Section 1.5 of this chapter has been published. Kaeli C. M. Johnson, Oliver X. Dong, Yan Huang and Xin Li. (2012) Cold Spring Harbor Symposia on Quantitative Biology 77: 259-68. 2  of chemical-resistant pathogens that are even more difficult to control. One of the aims of studying the endogenous defense mechanisms employed by plants is to provide insights that may aid in the development of sustainable solutions to modern agricultural challenges. The tiers of the plant immune system will be discussed in this chapter, with a particular emphasis on immunity mediated by NOD-like receptor (NLR) proteins.  1.2 Physical and chemical barriers  The first line of defense against pathogen attack includes physical and chemical barriers to infection. One proposed physical barrier is the plant cuticle, which coats the aerial tissues of land plants and is composed of a matrix of cutin and associated cuticular waxes. Although the primary role of the cuticle is to reduce water loss, a number of reports indicate that it also functions to limit microbial infection. Enhanced susceptibility to the fungal pathogen Exserohilium turcicum was observed in Sorghum bicolor mutants with altered cuticular structures (Jenks et al. 1994). Similarly, tomato mutants with severe cutin deficiencies in the fruit cuticle were shown to be more susceptible to infection by the necrotrophic pathogen Botrytis cinerea (Isaacson et al. 2009). The plant cell wall presents another physical barrier to infection, as the network of cellulose, hemicellulose, and pectin that forms the cell wall is resistant to physical penetration.  Counter-intuitively, mutant studies in Arabidopsis have revealed that defects in these physical barriers can lead to enhanced disease resistance. A leaky mutation in the cellulose synthase gene CESA3 (CELLULOSE SYNTHASE 3) results in enhanced resistance to powdery mildew which also correlates with a constitutive enhancement of jasmonate signaling (Ellis et al. 2002). From this work, it has been proposed that the cell wall is part of a mechanosensitive 3  signal transduction pathway and that modifications resulting in altered cell turgor may lead to defense response activation (Vorwerk et al. 2004). Relatedly, a number of Arabidopsis mutants with deficiencies in cutin biosynthesis display enhanced resistance to B. cinerea (Bessire et al. 2007; Chassot et al. 2007; Tang et al. 2007). The authors of these studies hypothesize that plants with more permeable cuticles are able to export antifungal compounds more readily, which is supported by the finding that leaf diffusates from mutants with altered cuticular structures have enhanced antifungal activity (Bessire et al. 2007). However, these findings may be an artifact resulting from the use of Potato Dextrose Broth in pathogen inoculation (Nawrath et al. 2013), as a study that performed water-based B. cinerea inoculations found that the cuticle development mutant glabra1 displayed enhanced susceptibility to this pathogen (Xia et al. 2010). Together, these findings highlight the complicated interplay between seemingly passive resistance structures and active, inducible immune responses; the latter will be discussed in more detail in the subsequent sections of this chapter. Chemical defense barriers include the production of anti-microbial enzymes and secondary metabolites. Plants produce a diverse array of defense-related chemicals with antibiotic activities through a variety of metabolic pathways. These compounds can be divided into two broad categories: phytoanticipins, which are constitutively produced, and phytoalexins, which are generated following pathogen detection (Van Etten et al. 1994).  Together these physical and chemical barriers block many attempts at infection, although some pathogens are able to circumvent these defenses. For example, upon detecting cutin the fungal pathogen Fusarium solani f.sp. pisi produces and secretes cutinase (Lin & Kolattukudy 1978). Many pathogenic fungi species possess a large number of cellulases, xylanases, and other cell wall degrading enzymes, the optimum activities of which are specific to their respective host 4  plants (King et al. 2011). Many microbes have evolved the ability to enzymatically detoxify certain plant defense compounds; Colletotrichum coccodes and Septoria lycopersici are able to degrade the toxic steroidal glycoalkaloid α-tomatine produced by Lycopersicum species, and this contributes to their ability to successfully parasitize the host plant (Sandrock & Van Etten 2001). When the physical and chemical barriers are breached, plants must rely upon their inducible defense responses to halt the progress of infection. The current conceptual understanding of the inducible plant immune system is based on the types of receptors and the resistance signaling pathways they initiate.  1.3 Immunity triggered by pathogen-associated molecular patterns  For the innate immune system to be effective, plants must have a sensitive, difficult to evade mechanism for detecting pathogen presence.  They possess a number of immune receptors with varied extracellular recognition motifs that localize to the cell surface; these proteins are termed pattern recognition receptors (PRRs), and can be classified as either receptor-like kinases (RLKs) or receptor-like proteins (RLPs) depending on whether or not they possess an intracellular serine/threonine kinase domain. Many PRRs recognize pathogen-associated molecular patterns (PAMPs), which are conserved features that are essential for the microbial lifestyle and include such things as the bacterial motility organ component flagellin and the fungal cell wall constituent chitin, amongst others (Boller & Felix 2009). Other PRRs detect damage-associated molecular patterns (DAMPs), which are signals indicative of pathogen-induced damage to the host cell and may include fragments of the plant cell wall or plant-derived peptides (Krol et al. 2010; Monaghan et 5  al 2012). Following ligand perception some PRRs associate with BAK1, an RLK first identified as a key component in brassinosteroid signaling (Li et al. 2002). The flagellin receptor FLS2 (FLAGELLIN SENSITIVE 2) and the DAMP receptors PEPR1 (PEP1 RECEPTOR 1) and PEPR2 are among the PRRs that form ligand-induced associations with BAK1 that are essential for downstream signal enhancement (Roux et al. 2011); however, other PRRs such as the chitin receptor CERK1 (CHITIN ELICITOR RECEPTOR KINASE 1) have downstream signaling pathways that are BAK1-independent (Gimenez-Ibanez et al. 2009).  Ligand perception by PRRs leads to the activation of PAMP-triggered immunity (PTI), which is initially characterized by an influx of calcium (Ma & Berkowitz 2007) and an increase in the production of reactive oxygen species (ROS) (O’Brien et al. 2012), which are early steps in the signaling pathways that lead to defense response outputs. PTI also leads to the activation of mitogen-activated protein kinase signaling cascades, resulting in large-scale transcriptional reprogramming that increases the expression of defense-related genes, the accumulation of phytoalexins, and the synthesis of defense-related phytohormones (Meng & Zhang 2013). While PTI is sufficient to halt the advances of most potential pathogens, some are able to overcome this type of immunity through the use of effector proteins which disrupt PTI signal transduction. Our understanding of the mechanisms underlying effector biology are largely based on studies performed using bacterial pathogens, which employ a syringe-like structure known as a type-III secretion system to inject effector molecules into the host cytoplasm (Cunnac et al. 2009). Pathogenic bacterial strains typically have a complement of 20-30 effector proteins that can vary widely between species and often have redundant functions within the plant cell. For example, Pseudomonas syringae encodes at least four effectors (AvrB, AvrRpm1, AvrRpt2, and HopF2) that target the Arabidopsis protein RIN4 (RPM1-INTERACTION PROTEIN 4) (Axtell 6  et al. 2003; Mackey et al. 2003; Wilton et al. 2010). Eukaryotic pathogens, including fungi and oomycetes, also secrete effector molecules that inhibit PTI into the cytoplasm of the cells of the host plant, but the method of delivery is still poorly understood (Rafiqi et al. 2012). Oomycete effectors typically contain a conserved RXLR motif that is necessary for translocation into the plant cell (Whisson et al. 2007); no such conserved motifs have been identified for fungal effector proteins.  1.4 Immunity triggered by effector recognition  1.4.1 Overview In an escalation of the “arms race” between plants and pathogens, plants have evolved an assemblage of intracellular receptor proteins that are able to recognize effector molecules either through direct protein-protein interactions or by perceiving effector activities within the cytosol. Effector proteins diverge extensively between species meaning that effector-triggered immunity (ETI) has an inherent degree of specificity that is unachievable in PTI, which relies upon the detection of features that typify entire classes of organisms.   1.4.2 The structure of NOD-like receptor (NLR) proteins The receptors that recognize pathogen effectors are called resistance (R) proteins, and can be divided into five classes based on structure and subcellular localization (Dangl & Jones 2001). The largest class is comprised of STAND (SIGNAL TRANSDUCTION ATPASES WITH NUMEROUS DOMAINS) P-loop ATPases that belong to the AAA+ superfamily (Leipe et al. 2004). These intracellular proteins possess a central nucleotide binding (NB) domain and a C-7  terminal leucine rich repeat (LRR) domain. In recent plant pathology literature, NB-LRR proteins are typically referred to as NLR proteins based on their structural similarity to mammalian NOD (NUCLEOTIDE-BINDING OLIGOMERIZATION DOMAIN)–like receptor (NLR) proteins. Despite aspects of structural conservation, the function of mammalian NLR proteins is actually more similar to that of plant PRRs, in that they recognize conserved microbial features rather than effector proteins (Li et al. 2015). Another striking difference is that NLR families in plants are hugely expanded compared to metazoans (Jacob et al. 2013).  NLR proteins can be further subdivided based on their N-terminal domains. Some possess a TIR (TOLL INTERLEUKIN RECEPTOR) domain and are thus referred to as TNLs, while others possess a CC (COILED-COIL) domain and are termed CNLs. While immune signaling in dicots employs both types of NLRs, monocots only possess CNLs. The reason for this difference is poorly understood, and is only one aspect of the extensive diversity observed in NLR complements both between and within plant species.  Some plant NLR proteins possess additional, non-canonical domains. For example, the Arabidopsis TNL protein CHS3 (CHILLING SENSITIVE 3) contains a C-terminal LIM domain (Yang et al. 2010). Another Arabidopsis TNL protein (RESISTANT TO RALSTONIA SOLANACEARUM 1; RRS1) has a C-terminal WRKY domain (Deslandes et al. 2002). These supplementary domains have been demonstrated to play unique roles in effector recognition.   1.4.3 Effector detection A number of models have been proposed to explain the varied mechanisms by which NLR proteins recognize their cognate effector molecules (Khan et al. 2016). In the direct interaction model, NLR proteins bind effectors through direct protein-protein interactions. These 8  interactions seem to occur via the LRR domain of the NLR, as indicated by mutational analyses of the Arabidopsis TNL RPP1 and the flax TNLs L5 and L6 (Krasileva et al. 2010; Ravensdale et al. 2012).  In most cases, an indirect interaction model provides a better fit with the experimental data. Two variants of such a model have been proposed: the guard model and the decoy model. In the guard model, an NLR protein detects changes in the abundance or modifications of a host protein that is targeted by pathogenic effectors. A specific and well-characterized example can be found in the relationship between RIN4 and the CNL proteins RPS2 (RESISTANT TO PSEUDOMONAS SYRINGAE 2) and RPM1 (RESISTANCE TO PSEUDOMONAS SYRINGAE PV. MACULICOLA) in Arabidopsis. RIN4 is a regulator of basal resistance (Kim et al. 2005; Liu et al. 2009), and is targeted by effectors from a variety of pathogens (Axtell et al. 2003; Mackey et al. 2003; Wilton et al. 2010). These modifications to RIN4 are perceived by RPS2 and RPM1, which subsequently initiate ETI.  The decoy model is similar, except that the host protein targeted by effectors and monitored by a(n) NLR protein(s) has no immune function other than to aid in triggering ETI (van der Hoorn & Kamoun 2008). Decoy proteins are structurally similar to virulence targets in the basal defense pathway. The Arabidopsis protein kinase PBS1 (AVRPPHB SUSCEPTIBLE 1) is an example of a decoy. The CNL protein RPS5 (RESISTANT TO PSEUDOMONAS SYRINGAE 5) associates with PBS1, and is activated upon its cleavage by the P. syringae effector AvrPphB (Ade et al. 2007). In keeping with the definition of a decoy protein, PBS1 has not been implicated in basal defense although the related protein kinase BIK1 (BOTRYTIS-INDUCED KINASE 1) does have a role in PTI and is also cleaved by AvrPphB (Zhang et al. 2010). 9  As an extension of the decoy model, evidence suggests that the non-canonical domains possessed by some NLR proteins may function as built-in decoys. The WRKY domain of the TNL protein RRS1 is acetylated by the R. solanacearum effector PopP2 and associates with the P. syringae effector AvrRps4 (Le Roux et al. 2015; Sarris et al. 2015). It remains to be shown whether this is a trend that extends to other non-canonical NLR proteins. An emerging trend in the field of plant pathology is the importance of NLR protein pairs in mediating ETI (Griebel et al. 2014). A classic example is the genetic and molecular relationship between RRS1 and its partner NLR protein RPS4 (Narusaka et al. 2009). In rice, the CNL proteins RGA4 (RESISTANCE GENE ANALOG 4) and RGA5 (RESISTANCE GENE ANALOG 5) physically and functionally interact in mediating resistance to M. oryzae (Cesari et al. 2014), and the Arabidopsis TNL protein CSA1 (CONSTITUTIVE SHADE-AVOIDANCE 1) is required for immunity mediated by its TNL protein partner CHS3 (Xu et al. 2015). Of note, in all three examples the NLR protein pairs are located genomically adjacent to one another, suggesting that the transcription of genes encoding paired NLR proteins may be intrinsically linked. The mechanisms by which effectors are perceived are varied and complex. However, all modes of recognition result in the same phenomenon: NLR protein activation and subsequent induction of immune signaling.  1.4.4 NLR protein activation It has been proposed that ETI signaling depends upon NLR proteins switching into an activated conformation. In the uninduced state the NB domain preferentially binds ADP (Maekawa et al. 2011), and the LRR domain forms intramolecular bonds with the rest of the protein, thereby 10  impeding intermolecular interactions necessary for signal transduction (Takken et al. 2006). The current model for protein activation states that following effector detection, the NLR protein undergoes a molecular shift that releases the inhibitory action of the LRR domain. This shift allows for nucleotide exchange at the NB domain such that ADP is replaced with ATP, and this is thought to result in an additional conformational change that allows for downstream signaling (Bonardi & Dangl 2012). However, the exact biochemical mechanism of plant NLR activation is unclear.  1.4.5 Downstream signaling The signaling pathways downstream of NLR protein activation are only partially characterized. Some NLR proteins localize to the nucleus and modulate defense-related gene expression. The barley CNL protein MLA10 (MILDEW A 10) associates with WRKY transcription factors in the nucleus to positively regulate immunity (Shen et al. 2007). This stands in contrast to the observed trend for many CNL proteins, which tend to localize to the plasma membrane and transduce immune signaling via the membrane-localized protein NDR1 (NON RACE-SPECIFIC DISEASE RESISTANCE 1) (Aarts et al. 1998; Takemoto et al. 2012). The tobacco TNL protein N associates with the transcription factor SPL6 (SQUAMOSA PROMOTER BINDING PROTEIN-DOMAIN TRANSCRIPTION FACTOR 6), and this association is necessary for resistance to tobacco mosaic virus (Padmanabhan et al. 2013). The Arabidopsis TNL protein SNC1 (SUPRESSOR OF NPR1, CONSTITUTIVE 1) associates with the transcription factor bHLH84 (BASIC HELIX-LOOP-HELIX 84) and its paralogs, which are positive regulators of immunity (Xu et al. 2014). 11  Signaling mediated by TNL proteins converges on the lipase-like protein EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1) and its interacting partners PAD4 (PHYTOALEXIN-DEFICIENT 4) and SAG101 (SENESCENCE-ASSOCIATED GENE 101) (Feys et al. 2001; Feys et al. 2005). EDS1 forms two distinct complexes with PAD4 and SAG101, respectively (Wagner et al. 2013), but the details of signaling through this node are still unclear.  The defense outputs of ETI are similar to those observed for PTI, although the induction of these responses is stronger, more rapid, and more robust during ETI (Tao et al. 2003; Tsuda & Katagiri 2010). Additionally, ETI is classically associated with a type of localized programmed cell death referred to as the hypersensitive response (HR), although a number of PAMPs have also been demonstrated to elicit HR (Bailey et al. 1990; Wei et al. 1992; Naito et al. 2008). The functional role of HR in plant immunity is somewhat contentious. It was previously posited that HR aided in containing the spread of pathogenic microbes. However, numerous studies have reported an uncoupling of cell death and resistance during immune signaling (Cole et al. 2001; Gassmann 2005; Hafez et al. 2012). As such, it is likely that the cell death associated with HR is a result (as opposed to a cause) of highly activated immune responses, as was first suggested over forty years ago (Kiraly et al. 1972).   1.5 Positive regulation of SNC1-mediated immunity  1.5.1 Overview In the snc1 gain-of-function mutant, a glutamic acid to lysine substitution in the linker region between the NB and LRR domains results in constitutive activation of defense without pathogen 12  perception.  The autoimmune phenotypes of snc1 include dwarfed stature, increased levels of SA, constitutive expression of PR genes, and increased resistance against virulent pathogens such as the bacteria Pseudomonas syringae pv. maculicola (P.s.m) ES4326 and the oomycete Hyaloperonospora arabidopsidis (H.a.) Noco2 (Zhang et al. 2003). Like most TNLs, SNC1 signals through PAD4 and EDS1.  SNC1 resides in the RPP4 (RECOGNITION OF PERONOSPORA PARASITICA 4) cluster on chromosome 4 in the Columbia (Col-0) ecotype. SNC1 is homologous to RPP4 and RPP5, with over 70% similarity at the amino acid level. The majority of the sequence dissimilarity occurs within the LRR domain, as the TIR-NB domain of SNC1 is almost identical to that of RPP4 and RPP5. . In support of the hypothesis that SNC1 encodes a bona fide NLR protein, loss-of-function snc1 alleles do not exhibit enhanced disease susceptibility, unlike what is observed in the loss-of-function activated disease resistance (adr) mutants which encode “helper” NLRs (Bonardi et al. 2011). However, the cognate effector protein for SNC1 remains to be discovered. As the snc1 mutation activates resistance signaling pathways without producing HR lesions, this unique autoimmune mutant is a useful tool for dissecting signaling events surrounding TNL activation. Multiple mutagenesis strategies have been employed in snc1 suppressor screens, yielding a number of modifier of snc1 (mos) mutants. We originally hypothesized that the activated snc1 protein needs sophisticated downstream signaling components such as EDS1 and PAD4 to transduce the “danger alarm” in order to induce defense outputs. The suppressor screen should yield mostly positive regulators of the signaling pathway. In the primary screen, the M2 generations of ethyl methanesulfonate (EMS), T-DNA, or fast-neutron mutagenized snc1 and snc1 npr1 populations were screened for loss of snc1 dwarf 13  morphology under normal lab growth conditions (i.e. 22°C, 16-hr day/8-hr night, ~50% humidity).  To confirm that mutants isolated from the primary screen are indeed snc1 suppressors and are involved in plant immunity, defense-related phenotypes including PR gene expression, endogenous SA levels and resistance to P.s.m. ES4326 and H.a. Noco2 were assessed in the M3 populations as part of the secondary screen.  Consistent with the prediction, the snc1 mos mutants exhibit wild type-like morphology and have decreased levels of all examined defense outputs. In total, fifteen independent complementation groups of mutants exhibited varying degrees of suppression of the snc1 autoimmune phenotypes. During the secondary screen, one of the challenges we faced was to identify and discard mutants containing intragenic snc1 mutations, which constituted the majority of the mutants isolated from the primary screen. Fortunately, loss-of-function snc1 mutations typically result in a dominant wild type-like morphological phenotype when heterozygous with the gain-of-function snc1 mutation. Using this criterion, we were able to focus on the <10% of the recessive mutants carrying mutations in second-site genes. Positional cloning was used to clone 11 of the 13 novel MOS genes. To facilitate mapping, snc1, originally in the Col background, was introgressed into Ler through six backcrosses. Crosses between snc1 mos mutants and the Ler-snc1 line were generated. The F1 plants, which displayed snc1-like morphology due to the recessive nature of the mos genes, were allowed to self-fertilize to generate a segregating F2 population suitable for conducting linkage analysis between the suppression of snc1 morphology and molecular markers (Zhang and Li 2005). Typically, a population of 600 – 1000 F2 plants would enable us to narrow the region containing each mutation to less than 100 kb, and genes within that region were directly 14  sequenced to identify the putative molecular lesions. Once the mutations were identified, MOS gene cloning was confirmed by transgenic complementation and allelism tests if T-DNA alleles were available from the Arabidopsis Biological Resource Center (ABRC).  As expected, several mutant alleles of PAD4 were identified. To date 13 novel MOS genes have been cloned using positional cloning (11) and T-DNA tagging (2) approaches. These encode proteins involved in RNA processing, nucleocytoplasmic trafficking, protein modification and epigenetic control of gene expression. The diverse MOS functions suggest that the activation of NLR-mediated resistance is highly complex (Palma et al. 2005; Zhang and Li 2005; Zhang et al. 2005; Goritschnig et al. 2007; Palma et al. 2007; Wiermer et al. 2007; Goritschnig et al. 2008; Cheng et al. 2009; Germain et al. 2010; Li et al. 2010; Xu et al. 2011; Xu et al. 2012).  1.5.2 Regulators of SNC1 gene expression levels: MOS1 and MOS9 The snc1 mutant displays morphological dwarfism together with constitutive defense activation, suggesting that maintaining NLR protein-mediated defense requires sacrifices to plant fitness. Accordingly, NLR gene expression must be tightly controlled under normal conditions. However, the mechanisms regulating NLR gene transcriptional control are largely unknown. In addition to conventional transcriptional regulation by transcription factors, epigenetic regulation via chromatin or histone modification has emerged as important for fine-tuning transcriptional control. For example, expression of the TNL-encoding gene LAZ5 (LAZARUS 5) requires trimethylation of H3K36 (Histone 3, Lysine 36) by the histone lysine methyltransferase SDG8 (SET DOMAIN GROUP 8) in order to maintain a transcriptionally active chromatin state (Palma et al. 2010). 15  The identification of MOS1 and MOS9 has provided insights into the mechanisms regulating TNL-encoding gene expression. Like other mos mutants, loss of either MOS1 or MOS9 function suppresses all snc1-related phenotypes. MOS1 encodes a protein containing a HLA-B ASSOCIATED TRANSCRIPT 2 (BAT2) domain that is conserved in both plants and animals (Li et al. 2010). Although an alteration of DNA methylation in a region upstream of SNC1 is observed in the mos1 mutant, it is not correlated with the repression of snc1 expression level, thus MOS1 likely does not function directly in DNA methylation. Furthermore, transgenic snc1 expression is not altered in mos1, indicating that MOS1 may regulate SNC1 expression at its specific chromosomal location. Interestingly, the repression of snc1 expression level can be mitigated by introducing ddm1 (decrease in dna methylation 1), suggesting that MOS1 and DDM1 may function antagonistically to regulate the expression of SNC1 at the chromatin level. MOS9 encodes a plant-specific protein of unknown function (Xia et al. 2013). In the loss-of-function mos9 mutant, the expression levels of SNC1 and another TNL-encoding gene, RPP4, are reduced. Immunoprecipitation of MOS9 followed by mass spectrum analysis identified ATXR7 (ARABIDOPSIS TRITHORAX-RELATED 7) as a MOS9-associated protein.   ATXR7 is a SET domain-containing H3K4 (Histone 3, Lysine 4) methylase required for the proper transcriptional activation of FLC (FLOWERING LOCUS C) via bulk methylation (Tamada et al. 2009). Identification of ATXR7 as a protein associated with MOS9 suggests that MOS9 likely regulates NLR gene transcription through H3K4me3 chromatin modification.  1.5.3 Components of the RNA processing machinery: MOS2, MOS4, and MOS12 To ensure appropriate functionality, stability and localization, nascent pre-mRNA transcripts are subject to a number of processing steps including 5’ capping, 3’ polyadenylation, and splicing, 16  followed by nuclear export. From our MOS screens, three genes were identified as important elements in RNA processing. MOS2 is required for both CNL- and TNL-mediated resistance (Zhang et al. 2005). It is also required for basal resistance against P.s.m. ES4326. These results suggest that MOS2 acts as a point of convergence for a number of immune signaling pathways. MOS2 is a nuclear protein containing one G-patch and two KOW (Kyrpides, Ouzounis, Woese) motifs, which are conserved among the MOS2 homologs in higher plants and animals. The glycine-rich G-patch motif is often found proximate to RS and RGG amino acid repeats, which have been implicated in non-specific protein-RNA interactions (Aravind and Koonin 1999). Similarly, the KOW motif from the bacterial elongation factor NusG has demonstrated nucleic acid binding activity and shares structural homology with the tudor protein-protein interaction motif (Steiner et al. 2002). While MOS2 has not been experimentally shown to bind RNA, the human MOS2 homolog GPKOW binds RNA in a manner dependent on its phosphorylation by protein kinase A (Aksaas et al. 2011). Furthermore, the remote yeast homolog SPP2 (SPLICEOSOMAL PROTEIN 2) is an essential protein required for the first RNA cleavage step in pre-mRNA splicing as the G-patch domain of SPP2 associates with  Prp2 (PRE-mRNA PROCESSING 2), an RNA-dependent ATPase that activates the spliceosome (Roy et al. 1995; Silverman et al. 2004; Yeh et al. 2011).  Therefore, MOS2 is predicted to bind RNA, appears to function in splicing, and may also interact with other proteins via its KOW motif. However, the precise role of MOS2 in RNA processing, particularly within an immunity context, remains unclear. Interestingly, the involvement of the yeast MOS2 homolog in pre-mRNA splicing provides evidence that a connection may exist between the roles of MOS2 and MOS4 in regulating plant immunity.  17  Like MOS2, MOS4 is a nuclear protein that is required for basal resistance against P.s.m. ES4326, as well as for both CNL- and TNL-mediated resistance (Palma et al. 2007). Along with 23 other proteins it forms a highly conserved spliceosome-associated complex known as the MOS4-associated complex (MAC) in Arabidopsis (Monaghan et al. 2009). All examined MAC component single mutants are viable but display pleiotropic defects, while all double mutant combinations are lethal (Nemeth et al. 1998; Palma et al. 2007; Monaghan et al. 2009; Monaghan et al. 2010). This indicates that while individual MAC components may be involved in regulating a number of different biological processes, the complex as a whole is required for some essential function, such as general mRNA splicing.  The yeast and human orthologous complexes have been implicated in spliceosome assembly and pre-mRNA splicing (Tarn et al. 1993; Ajuh et al. 2000; Ohi et al. 2002; Deckert et al. 2006), and it is expected that the MAC plays a similar role in plants due to the conserved nature of the complex. Several MAC components, including MOS4, were recently shown to be necessary for the proper splicing of RPS4 and SNC1 (Xu et al. 2012). It is tempting to hypothesize that defense-related gene transcripts may be differentially processed upon pathogen detection through modulation of the MAC. While MOS2 has not been shown to directly associate with the MAC, the human and yeast homologs of MOS2 have been implicated as components of the conserved spliceosome-associated complex (Roy et al. 1995; Bessonov et al. 2010; Aksaas et al. 2011). Additionally, MOS2 has been shown to be required for proper splicing of SNC1 (S. Xu and Y. Zhang, unpublished data). This provides further support that MOS2 is also involved in RNA processing, possibly in conjunction with the MAC. 18  MOS12 is required for basal resistance against P.s.m. ES4326, as well as for resistance mediated by a subset of NLR proteins, primarily those belonging to the TNL class (Xu et al. 2012). It encodes a nuclear arginine-rich protein with two cyclin domains at the N-terminus. Its closest homolog is human cyclin L, which is predicted to be involved in mRNA splicing due to its association with splicing factors and ability to stimulate splicing in vitro (Dickinson et al. 2002; de Graaf et al. 2004). The mos12 allele isolated from the MOS screens (mos12-1) contains a point mutation at an intron-exon splice junction that causes a reading frame shift, resulting in a truncated protein (Xu et al. 2012). However, the truncated protein is likely still partially functional as the null mos12-2 T-DNA insertion allele is lethal, indicating that this gene plays an essential role in plant growth and development. In mos12-1 plants, the splicing patterns of both SNC1 and RPS4 are altered from those observed in wild type plants (Xu et al. 2012). In addition, MOS12 co-immunoprecipitates with MOS4. Together, these results indicate that MOS12 plays a critical role in the splicing of NLR gene transcripts, likely in association with the MAC through the spliceosome. Plant defense responses may be regulated in part through alternative splicing of NLR gene transcripts, as pathogen detection has been shown to elicit the production of splice variants of a number of TNL-encoding genes, including tobacco N and Arabidopsis RPS4 (Dinesh-Kumar and Baker 2000; Zhang and Gassmann 2003). In tobacco, transcripts of N are alternatively spliced following pathogen attack, and a specific ratio of full-length and truncated N proteins is thought to be required for complete resistance against tobacco mosaic virus as neither splice variant is able to individually induce defense response outputs (Dinesh-Kumar and Baker 2000). Similarly, alternative splicing of RPS4 is induced in Arabidopsis following pathogen detection, and 19  alternatively spliced transcripts are required for complete RPS4-mediated immunity (Zhang and Gassmann 2003). Increased expression of the RPS4 transcript is induced by the recognition of avrRps4, HopA1, or avrRpt2 effector molecules, of which only avrRps4 is specifically recognized by RPS4 (Zhang and Gassmann 2007). Additionally, detection of avrRps4 resulted in the altered splicing of not only RPS4 but also of two other Arabidopsis genes known to have alternatively spliced forms, only one of which is thought to potentially function in defense response. Therefore, it has been proposed that increased TNL transcript production and alternative splicing may constitute a general response used to prime plants for resistance. While alternative splicing of R genes has been predominantly observed for TNL transcripts, there are reports of CNL gene transcript alternative splicing as well (Halterman et al. 2003; Peart et al. 2005).    1.5.4 Nuclear proteins important for mRNA export: The Nup107-160 complex and MOS11 Cellular compartmentalization in eukaryotic cells requires that processed transcripts be delivered into the cytoplasm before translation can occur. This provides an additional tier of regulation by controlling the nuclear export of mature mRNA molecules. Export of transcripts that are successfully processed requires RNA export proteins such as nuclear export factors, nucleoporins, and RNA chaperones. Although much insight has been gained using human and yeast models, RNA nucleocytoplasmic trafficking is still poorly understood in plants. Common features exist in the mos3 and mos11 mutants, as they both suppress the constitutive autoimmune phenotypes of snc1 (Zhang and Li 2005; Germain et al. 2010). In addition, the mos3 single mutant exhibits enhanced disease susceptibility to both virulent and 20  avirulent pathogens, suggesting the role of MOS3 in both basal defense and NLR protein-mediated defense. However, such increased susceptibility was not observed in the mos11 single mutant. In situ hybridization of total mRNA revealed a dramatic accumulation of transcripts in the nucleus of each mutant, which suggests that MOS3 and MOS11 are both required for successful mRNA export (Parry et al. 2006; Germain et al. 2010).  MOS3 localizes to the nuclear rim while MOS11 is present in the nuclear matrix, suggesting that MOS11 may function before MOS3 in the mRNA export process (Zhang and Li 2005; Germain et al. 2010). MOS11 encodes a homolog of human CIP29, an RNA co-chaperone enhancing the activity of RNA helicase DDX39 (Sugiura et al. 2007; Dufu et al. 2010). MOS11 may function as part of a similar complex during the mRNA export process in plants. MOS3 is homologous to NUCLEOPORIN (Nup) 96 in mammals and Nup145 in yeast, both of which have been reported to be involved in mRNA export (Fabre et al. 1994; Vasu et al. 2001). Nup96 functions as part of the Nup107-160 nuclear pore sub-complex. In mice, the loss-of-function nup96 allele is lethal when homozygous and the immune systems of heterozygotes are severely impaired, indicating that Nup96 functions in both innate and adaptive mammalian immunity (Faria et al. 2006). MOS3 is thought to function as part of a homologous complex in Arabidopsis, and other putative complex components, including Nup160 and Seh1, were recently shown to be  required for basal and TNL-mediated resistance (Wiermer et al. 2012). While it is uncertain how a general defect in non-specific mRNA export impairs NLR-mediated immunity without serious developmental consequences, one possibility is that plants have evolved to be more resilient, and loss of only one component of the Nup107-160 complex does not lead to lethality. However, the double mutant of nup96 nup160 is seedling lethal (Parry et al. 2006).  21  1.5.5 Components involved in nucleocytoplasmic protein trafficking: MOS6, MOS7, and MOS14 The mos6 mutant alleles were identified in the suppressor screens either in snc1 or snc1 npr1 backgrounds (Palma et al. 2005). In all cases, mos6 alleles partially suppress the autoimmune phenotypes of snc1. Interestingly, the mos6 single mutant exhibits enhanced disease susceptibility to H.a. Noco2, but not to P.s.m. ES4326, indicating that MOS6 may play a specific role in basal defense against oomycete infection. Positional cloning of MOS6 showed that it encodes importin α3. The MOS6 protein has the typical features of importin α proteins, including an importin β-binding domain and nuclear localization signal (NLS)-binding pockets. GFP-localization of MOS6 showed that it is concentrated in the nucleus, which supports the idea that it is a functional importin α (Palma et al. 2005). Within the Arabidopsis genome, there are eight genes encoding homologs of importin α, including MOS6. This large number of importin homologs in Arabidopsis is unsurprising, given the essential role of protein import in every aspect of plant growth and development. Whether a subset of these importin α homologs is specifically involved in plant defense response is not known. Since all importin α proteins bind NLS, there are likely protein-protein interaction domains defining specificity.  Besides its involvement in snc1-mediated defense, MOS7 has also been suggested to play roles in basal defense and defense mediated by other NLR proteins. Furthermore, SAR is compromised in the mos7 single mutant, as pre-treatment of the mos7 plants with an avirulent bacterial pathogen failed to trigger subsequent immunity in distal leaves (Cheng et al. 2009). MOS7 encodes a protein with homology to Nup88 in animals, which is involved in nuclear protein export (Uv et al. 2000). MOS7 localizes to the nuclear rim, indicating its potential role as a nucleoporin in Arabidopsis. Intriguingly, the nuclear accumulations of NPR1, EDS1 and SNC1 22  are reduced in the mos7-1 single mutant as compared to wild type, while the nuclear distributions of HDA19, CDC5 and TGA2 remain unchanged (Cheng et al. 2009). This suggests that there is specificity in the protein export pathways affected by the mos7-1 mutation. However, since MOS7 is a single-copy gene in the Arabidopsis genome and the mos7-2 null T-DNA insertion allele is lethal, wild type MOS7 is likely required for general nuclear protein retention. Further work is required to determine how the mos7-1 mutation leads to the specific enhancement of nuclear export activity of immunity-related proteins such as SNC1, EDS1, and NPR1. MOS14 is a required intermediate in both basal resistance and some TNL-mediated resistance signaling pathways (Xu et al. 2011). It is a single copy gene in Arabidopsis encoding a nuclear protein with homology to metazoan transportin-SR (TRN-SR) proteins, which are nuclear import receptors. TRN-SR proteins belong to the importin-β super-family, members of which mediate the import of protein cargo through the nuclear pore complex (NPC) upon recognition of an NLS. TRN-SR proteins specifically transport serine-arginine rich (SR) proteins, which function in both constitutive and alternative splicing through their roles in pre-mRNA splice site recognition and spliceosome assembly (Long and Caceres 2009).  The MOS14 protein was shown to interact with four different SR proteins through its C terminus and AtRAN1, which is required for release of the cargo into the nucleus, through its N terminus (Xu et al. 2011). In the homozygous mos14-1 mutant, nuclear localization of SR proteins is impaired. In keeping with this mislocalization of known splicing factors, the splicing patterns of SNC1 and RPS4 are altered in mos14-1 plants, and resistance mediated by these proteins is attenuated. Studies of MOS6, MOS7, and MOS14 have revealed the importance of nucleocytoplasmic protein trafficking in the regulation of plant defense. In addition to the mRNA 23  export process discussed in previously, fine-tuned nuclear import and export of defense regulators seem to play a key role in mounting effective immunity in plants.  1.5.6 Transcriptional co-repression with SNC1: MOS10 (TPR1)  MOS10 encodes a nuclear protein with high sequence similarity to TOPLESS (TPL) (Zhu et al. 2010), a transcriptional corepressor that functions during embryogenesis in an auxin-dependent manner (Szemenyei et al. 2008). As such, MOS10 was renamed TOPLESS RELATED 1 (TPR1). Like TPL, TPR1 is a transcriptional corepressor, which is a unique biological function amongst the identified MOS proteins. Both TPR1 and TPL are required for basal immunity, as well as resistance mediated by a number of TNL proteins (Zhu et al. 2010). Overexpression of TPR1 results in the constitutive activation of defense phenotypes similar to those observed in the snc1 mutant, including increased SA accumulation, constitutive PR gene expression, and enhanced resistance to H.a. Noco2. These responses in the TPR1 overexpression lines require EDS1, PAD4, and SNC1. Co-immunoprecipitation experiments showed that TPR1 and SNC1 associate with one another in planta, likely through the TIR domain of SNC1. The homozygous tpl mutant displays phenotypes similar to those observed in histone deacetylase 19 (hda19), including apical shoot defects (Long et al. 2006). Additionally, the hda19 single mutant exhibits compromised pathogen resistance (Kim et al. 2008). Co-immunoprecipitation experiments indicate that TPR1 associates with HDA19 in vivo (Zhu et al. 2010). Histone deacetylases remove acetyl groups from a histone lysine residue, thereby enhancing DNA condensing which in turn inhibits transcription. TPR1 has been shown to associate with the promoters of DEFENSE NO DEATH 1 (DND1) and DND2, two known 24  negative regulators of immunity (Yu et al. 1998; Yu et al. 2000), and may act together with HDA19 to regulate their transcription. SNC1 and other NLR proteins may activate downstream defense responses in part by modulating the transcriptional repression activity of TPR1.   1.5.7 Protein modifying enzymes: MOS5 and MOS8 In most eukaryotes, post-translational modifications (PTMs) modulate protein function by influencing their activity, stability and localization. PTMs are needed to regulate a diverse range of cellular functions. Increasing evidence indicates that PTMs, such as ubiquitylation and phosphorylation, play an important role in plant defense signaling. The identification of MOS5 and MOS8 indicates that PTMs are crucial in regulating NLR protein activation. MOS5 encodes one of two ubiquitin-activating (E1) enzymes in Arabidopsis (Goritschnig et al. 2007).  Along with E2 (Ubiquitin conjugating enzyme) and E3 (Ubiquitin ligase) enzymes, E1s are involved in labeling protein substrates with ubiquitin moieties, typically to mark a substrate for degradation by the 26S proteasome. The loss of MOS5 function partially suppresses snc1 phenotypes and leads to both impaired basal and NLR--mediated defense activity. The single mutant of the other Arabidopsis E1 enzyme, uba2, displays no obvious phenotypic defects, but the mos5 uba2 double mutant is lethal, indicating that a large degree of redundancy exists between these two E1 enzymes.  The mos5 mutant contains a molecular lesion in the putative ubiquitin-fold domain, which likely disrupts the ubiquitylation process. The enhanced susceptibility phenotypes observed in the mos5 mutant may result from the increased stability of negative defense regulators, the degradation of which might be essential in snc1-mediated defense resistance. 25  Alternatively, the mos5 mutation may disrupt the function of positive defense regulators, which may require mono-ubiquitination for activation. Recent work has shown that the F-box protein CPR1, which  belongs to the SKP1-CULLIN1-F-box (SCF) E3 complex, targets SNC1 and other NLRs for degradation, highlighting the importance of ubiquitylation in regulating NLR protein levels and preventing autoimmunity (Cheng et al. 2011). Surprisingly, while mos5 suppresses the dwarf phenotype of snc1, it enhances the stunted growth morphology observed in cpr1-2 (Gou et al. 2012). The complex mos5 phenotype is likely a result of the mutant allele’s impact on multiple E3 enzyme activities (Cheng and Li 2012).  Other PTMs that modify protein localization, solubility, and protein-protein interactions are also required in defense signal transduction. The common lipid modification, prenylation, involves the covalent binding of hydrophobic farnesyl- or geranylgeranyl- moieties to the target proteins, likely facilitating their binding to cellular membranes (Galichet and Gruissem 2003). mos8 is an allele of ERA1 (ENHANCED RESPONSE TO ABSCISIC ACID 1), which encodes the β-subunit of farnesyltransferase (Goritschnig et al. 2008). Like other era1 alleles, mos8 displays enhanced susceptibility to P.s.m. ES4326 and H.a. Noco2, implying a role for farnesylation in basal immunity. It also exhibits impaired defense responses mediated by several NLR proteins. Furthermore, epistatic analyses using era1 and several abscisic acid (ABA) biosynthesis mutants indicates that enhanced susceptibility of era1 is only partially dependent on ABA. The era1 npr1 double mutant displays enhanced era1 phenotypes, indicating that ERA1 functions in an NPR1-independent pathway. Even though the target proteins modified by ERA1 are currently unknown, lipid modification is likely playing an important role in disease resistance signaling by targeting substrate proteins to cellular membranes and altering their activities.  26  1.5.8 Integration of the MOS genes The results of the MOS screens were somewhat surprising in that they primarily resulted in the identification of novel factors regulating SNC1 expression, RNA processing, nucleocytoplasmic trafficking, and protein localization and activity of NLR genes and their encoded products. We propose a model that brings together these seemingly disparate functions within a resistance signaling context, centering on NLR protein activation (Figure 2.1).   Figure 1.1. A model depicting the involvement of the MOS proteins in NLR protein-mediated defense signaling pathways in Arabidopsis, using SNC1 as an example of the journey of TNL proteins. 1. At chromosomal level, MOS1, ATXR7 and MOS9 up-regulate the transcription of SNC1 through chromatin remodeling. 2. MOS2, MOS4, and MOS12 are required for the proper 27  splicing of the transcripts of SNC1. 3. The Nup107-160 complex and MOS11 play key roles in the export of total mRNA (including mature mRNA of SNC1), which is required for effective defense. 4. MOS5 is an E1 ubiquitin-activating enzyme, an essential component of the ubiquitination cascade, required for the regulation of defense signaling components. As an example, the SCFCPR1 E3 ubiquitin ligase complex targets SNC1 for degradation which prevents autoimmunity caused by over-accumulation of NLR proteins. MOS8 positively regulates plant defense, possibly through prenylation that affects the targeting of defense regulators. 5. MOS6 and MOS7 are involved in the nucleocytoplasmic shuttling of defense signaling molecules such as SNC1, EDS1, and NPR1. Like with RPS4, EDS1 is probably required for the nuclear localization and activation of SNC1 upon the recognition of its corresponding effector (Bhattacharjee et al. 2011; Heidrich et al. 2011). MOS14 is required for the nuclear import of splicing factors that may affect defense regulator RNA processing. 6. MOS10 activates the SNC1-mediated defense through transcriptional repression of negative regulators of defense such as DND1 and DND2.  Our model suggests that NLR protein-mediated signaling may not consist of a long, linear signaling pathway possessing numerous intermediates as we imagined earlier. Rather, the primary regulatory steps exist at the gene and protein processing levels. Collectively, the characterized MOS genes provide new insights into the complex regulatory mechanisms governing NLR-mediated immunity.  In the absence of pathogens, low NLR gene expression maintains a small reservoir of NLR proteins which putatively act as a surveillance system, protecting against pathogen attack. Upon pathogen detection, a number of factors including MOS1, ATXR7 and MOS9 likely alter chromatin structure or histone codes in order to up-regulate NLR gene transcription, thus enhancing pathogen detection capabilities and amplifying defense response signaling (Li et al. 2010; Y. Cheng and X. Li, unpubli.).  The processing of NLR gene transcripts is another regulatory node. MOS2, MOS4, MOS12 and MOS14 are all required for correct NLR transcript splicing (Xu et al. 2011; Xu et al. 2012; F. Xu and Y. Zhang, unpubl.). Following pathogen detection, these proteins may aid in recruiting the spliceosome preferentially to NLR gene transcripts in order to increase the speed, 28  specificity and strength of defense response activation. Alternatively, these proteins may play a role in NLR gene transcript alternative splicing, which is thought to provide specificity in the type and strength of immune response activated. After splicing, transcripts must be exported to the cytosol to be translated.  The Nup107-160 complex, which includes MOS3, functions in the same mRNA export pathway as MOS11 (Zhang and Li 2005; Germain et al. 2010; Wiermer et al. 2012). The mRNA export process could be another regulatory step modulating the final defense response outputs from NLR genes. After translation in the cytosol, protein activity and localization may be altered by PTMs. MOS5 functions as an essential part of the ubiquitination pathway, which results in the addition of ubiquitin moieties to target proteins (Goritschnig et al. 2007). This may mark negative defense regulators for proteasomal degradation or, alternatively, activate positive regulators. MOS5 likely has a general role in ubiquitination. MOS8 is required for prenylation (Goritschnig et al. 2008), a type of PTM that regulates protein membrane targeting (Resh 2006). MOS8 may direct defense regulators to their correct locations thereby allowing signal transduction to proceed, although its targets have not yet been identified. Following protein synthesis and modifications, many defense regulators are transported back to the nucleus in order for defense activation to occur. MOS6 and MOS7 are likely involved in the nuclear import and retention of defense-related proteins, although the degree of specificity in the activity of these proteins is unclear (Palma et al. 2005; Cheng et al. 2009). MOS14 is required for the nuclear import of SR proteins, most of which are splicing factors (Xu et al. 2011). While MOS14 is required for precise NLR gene splicing, specificity has not been demonstrated. Thus, NLR-mediated immunity may also be regulated by factors involved in 29  nuclear import and export of proteins. These events affect the nuclear retention of key defense regulators and in turn determine the final levels of defense outputs. MOS10/TPR1 is unique among the MOS proteins. It represses the transcription of known negative regulators of defense and associates with SNC1 in the nucleus, likely as part of a complex (Zhu et al. 2010). The association of SNC1 with MOS10/TPR1 appears to be required for the strong and rapid repression of negative defense regulators in order to mount an effective defense response. During the past decade, studies of the MOS genes in our laboratory have provided insight into the molecular details surrounding NLR-mediated immunity. However, the full picture of the regulation of NLR genes and their encoded products has not yet emerged.  The improved next generation sequencing strategies and biochemical approaches guarantee an impending revolution in molecular studies of plant biology.  1.6 Thesis objectives  The primary objective of the research presented in this thesis was to identify novel regulators of plant innate immunity. As the MOS screen previously conducted in the Li laboratory was highly successful in identifying positive regulators of NLR-mediated immunity by looking for suppressors of the autoimmune mutant snc1, an enhancer screen was utilized to identify negative regulators. This thesis describes the identification and characterization of three MUTANT, SNC1-ENHANCHING (MUSE) mutants isolated from this screen: muse9, muse4, and muse7. The aim of performing functional studies of these MUSE genes and the proteins they encode is to improve our understanding of the mechanisms that underlie plants’ endogenous defense responses. 30  Chapter 2: The chromatin remodeler SPLAYED negatively regulates SNC1-mediated immunity2  2.1  Summary  SNC1 (SUPPRESSOR OF NPR1, CONSTITUTIVE 1) is one of a suite of intracellular Arabidopsis NOD-like receptor (NLR) proteins which, upon activation, result in the induction of defense responses. However, the molecular mechanisms underlying NLR activation and the subsequent provocation of immune responses are only partially characterized. To identify negative regulators of NLR-mediated immunity, a forward genetic screen was undertaken to search for enhancers of the dwarf, autoimmune gain-of-function snc1 mutant. To avoid lethality resulting from severe dwarfism, the screen was conducted using mos4 (modifier of snc1, 4) snc1 plants, which display wild-type-like morphology and resistance. M2 progeny were screened for mutant, snc1-enhancing (muse) mutants displaying a reversion to snc1-like phenotypes. The muse9 mos4 snc1 triple mutant was found to exhibit dwarf morphology, elevated expression of the pPR2-GUS defense marker reporter gene, and enhanced resistance to the oomycete pathogen Hyaloperonospora arabidopsidis Noco2. Via map-based cloning and Illumina sequencing, it was determined that the muse9 mutation is in the gene encoding the SWI/SNF chromatin remodeler SYD (SPLAYED), and was thus renamed syd-10. The syd-10 single mutant has no observable alteration from wild-type-like resistance, although the syd-4 T-DNA insertion allele displays enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326.                                                  2 A version of this chapter has been published. Kaeli C. M. Johnson, Shitou Xia, Xiaoqi Feng and Xin Li. (2015) Plant and Cell Physiology 56(8):1616-23. 31  Transcription of SNC1 is increased in both syd-4 and syd-10. These data suggest that SYD plays a subtle, specific role in the regulation of SNC1 expression and SNC1-mediated immunity. SYD may work with other proteins at the chromatin level to repress SNC1 transcription; such regulation is important for fine-tuning the expression of NLR-encoding genes to prevent unpropitious autoimmunity.    2.2 Introduction  To compensate for the vulnerability inherent in being sessile organisms, plants must maintain a tightly regulated innate immune system to ward off pathogenic infection (Dangl et al. 2013). As part of this system, the detection of conserved microbial features by receptors on the plant cell surface induces relatively mild defense responses (Macho and Zipfel 2014). However, successful pathogens are able to deliver effector molecules into the host cell to suppress this immune response and promote infection.  As an additional line of defense, plants possess a suite of intracellular receptors termed RESISTANCE (R) proteins which recognize effectors in a specific manner either directly or through their effects upon other host proteins (Chisholm et al. 2006; Dangl and Jones 2001). Although there are several classes of R proteins, the majority belong to the nucleotide-binding and leucine-rich repeat domain-containing/NOD-like receptor (NLR) class. Upon effector detection, NLR proteins become activated and strong, robust defense responses are induced. NLR protein-mediated immunity is characterized by an accumulation of the defense hormone salicylic acid (SA), increased expression of PATHOGENESIS-RELATED (PR) defense marker genes, and often a programmed cell death event known as the hypersensitive response (HR) 32  (Hammond-Kosack and Jones 1996). While NLR-mediated immunity is a well-documented phenomenon in higher plants, the molecular mechanisms underlying its regulation are only marginally understood.  In the absence of pathogen attack, NLR protein levels must be kept under stringent control in order to prevent growth defects and potential lethality resulting from unwanted activation of autoimmune responses. Upon infection, however, the repression of NLR protein-mediated signaling pathways must be released in order to allow the rapid induction of defense responses. The regulation of NLR-mediated immunity occurs at the transcriptional, translational, and post-translational levels. At the transcriptional level, a number of positive regulators of NLR gene expression have been identified. The histone lysine methyl transferase SDG8 trimethylates H3K36 (Histone 3, Lysine 36) at the NLR-encoding LAZ5 locus, and this activity is required for the perpetuation of a transcriptionally active chromatin state (Palma et al. 2010). Similarly, MOS9 was shown to function together with the methyl transferase ATXR7 in the methylation of H3K4 at the NLR-encoding SNC1 and RPP4 loci, and this methylation is required for the full expression of these genes (Xia et al. 2013). The MOS1 protein, which contains an HLA-B ASSOCIATED TRANSCRIPT 2 domain, is required for full SNC1 expression and functions antagonistically with the chromatin remodeling factor DECREASED DNA METHYLATION 1 (DDM1) (Li et al. 2010; Li et al. 2011). Although the mechanism of this regulation is not well understood, it is thought to occur at the chromatin level as the expression of transgenic SNC1 does not require MOS1. MOS1 and MOS9 were both identified from a forward genetic screen designed to isolate positive regulators of NLR-mediated immunity. The MODIFIERS OF SNC1 (MOS) screen was designed to identify suppressors of the autoimmune mutant snc1 (suppressor of npr1, 33  constitutive 1), which contains a gain-of-function mutation in an NLR-encoding gene (Li et al. 2001; Zhang et al. 2003). Mutant snc1 plants display a dwarfed, dark green, curled-leaf morphology, accumulate SA, and exhibit constitutively activated defense responses, although lesions typically associated with HR fail to form. As such, the snc1 mutant has become a useful genetic background in which to conduct forward genetic screens for regulators of immunity. From the MOS screens, mutants exhibiting a suppression of snc1-mediated defense responses were selected and many mos mutations were cloned.  As the MOS screens were successful in identifying positive regulators of NLR-mediated immunity (summarized in Johnson et al. 2012), we proceeded to design enhancer screens in the snc1 background in order to identify negative regulators of immunity. To avoid lethality resulting from dramatic dwarfism the forward genetic screens were conducted by mutagenizing seeds from mos4 snc1 plants, which are wild-type-like in terms of morphology and resistance levels. As part of the MUTANT, SNC1-ENHANCING (MUSE) screen a number of mutants displaying a reversion back to snc1-like morphology and defense outputs were isolated, several of which have been recently published (Huang et al. 2013; Huang et al. 2014a; Huang et al. 2014b; Xu et al. 2015, in press). This study focuses on the isolation, identification, and characterization of muse9. The muse9 mos4 snc1 triple mutant is dwarfed and displays elevated expression of the pPR2-GUS reporter gene. An elevation in resistance against the virulent oomycete strain Hyaloperonospora arabidopsidis (H.a.) Noco2 was observed in the triple mutant. The muse9 mutation was found to be a novel allele of splayed (syd-10), which encodes a SWI/SNF chromatin remodeler. The syd-10 single mutant exhibits wild-type-like resistance, but the syd-4 T-DNA insertion allele exhibits enhanced resistance to Pseudomonas syringae pv. maculicola (P.s.m.) ES4326. Double mutant 34  analysis showed that mutations in the SYD locus enhance the dwarfism of snc1, and SYD is required for modulating transcription at the SNC1 locus. Thus, we establish that SYD plays a subtle but specific role in repressing SNC1 expression.   2.3 Results  2.3.1 Isolation of muse9 snc1 mos4 The muse9 mutation was isolated from the MUSE forward genetic screen described previously (Huang et al. 2013), which was conducted in the mos4 snc1 mutant background with ethyl methanesulfonate (EMS) as a mutagen. Mutant lines displaying a reversion to snc1-like phenotypes were selected as putative snc1 enhancers. The muse9 mos4 snc1 triple mutant displays snc1-like morphological phenotypes (Figure 2.1A).  In snc1, a number of PATHOGENESIS-RELATED (PR) defense marker genes are constitutively expressed. All mutants from the MUSE screens contain a reporter gene construct in which the promoter of PR2 is fused to the coding region of β-glucuronidase (GUS), allowing for a rapid visualization of defense gene expression. In the wild-type Columbia (Col-0) background no GUS expression is observed (Figure 2.1B). The muse9 mutation partially rescues the constitutive expression of the pPR2-GUS reporter gene observed in snc1 but suppressed in mos4 snc1 (Figure 2.1B).  The snc1 mutation confers enhanced resistance against the virulent oomycete pathogen H.a. Noco2 (Zhang et al. 2003). Consistent with the observed rescue of pPR2-GUS constitutive expression noted above, the muse9 mos4 snc1 triple mutant showed a moderate but significant enhancement in resistance against H.a. Noco2 as compared to the mos4 snc1 double mutant 35  (Figure 2.1C). Together, these data indicate that the muse9 mutation is able to partially enhance snc1 phenotypes in the mos4 snc1 background.  Figure 2.1. Phenotypic analysis of the muse9 mos4 snc1 triple mutant.  (A) Morphology of soil-grown Col-0, snc1, mos4 snc1, and muse9 mos4 snc1 plants. Photographs were taken 3 weeks post-germination. Scale bar indicates 1 cm. (B) PR2 gene expression depicted using the pPR2-GUS fusion construct present in all shown genetic backgrounds. Plants were grown for 10 d on MS media.   (C) Growth of H.a. Noco2 on indicated genotypes 7 d post-inoculation with 1x105 spores/mL. Values represent the average of 4 replicates of 5 plants each ± SD. Significant difference between mos4 snc1 and muse9 mos4 snc1 indicated by * (P-value < 0.05). The experiment was repeated three times with similar results.  2.3.2 Phenotypes associated with muse9 result from a point mutation in SYD To determine the molecular lesion responsible for the snc1-enhancing phenotypes associated with muse9, a positional cloning strategy was employed. The muse9 mos4 snc1 triple mutant in 36  the Col-0 ecotype was crossed with Landsberg erecta (Ler) to generate the mapping population. Linkage analysis was performed using 24 F2 plants displaying snc1-like phenotypes, which revealed that muse9 showed linkage with markers located between 9.2MB and 13.2MB on chromosome 2 (Figure 2.2A).  Fine mapping using >1000 F3 plants from F2 progeny that were homozygous for snc1 and MOS4, but heterozygous for muse9, further narrowed down muse9 to a region between 10.8MB and 12.4MB on chromosome 2.  To identify the exact mutation responsible for muse9, Illumina whole genome sequencing was performed. Comparisons between the mutant sequence and the reference Col-0 Arabidopsis genome indicated that five genes located within this mapped region contained mutations consistent with EMS mutagenesis (Figure 2.2B). However, four of these are either silent or intronic mutations. The other mutation is in At2g28290, and results in an amino acid change; therefore, it was selected as the most likely candidate for muse9. At2g28290 encodes SPLAYED (SYD), a SWI/SNF chromatin remodeling ATPase previously implicated in development as well as jasmonate (JA) and ethylene (ET) signaling pathways (Wagner and Meyerowitz 2002; Walley et al. 2008). The C to T substitution in muse9 occurs in the last exon of SYD in a region of the protein that does not contain any known conserved domains (Figure 2.2C-D), and results in the substitution of Ala2224 with Val (Figure 2.2E).  Transgene complementation is commonly employed in verifying positional cloning results. However, the large size of the SYD locus (>16 kb) precludes straightforward molecular cloning in binary plasmid vectors. Instead, to verify that the mutation in SYD is responsible for the muse9 phenotypes, an allelism test was carried out between the muse9 single mutant and syd-37  4, a previously published T-DNA insertion allele (Zhu et al. 2013) that contains an insertion in the conserved helicase domain of SYD (Figure 2.2D).   Figure 2.2. Positional cloning of the MUSE9 locus on chromosome 2. (A) A genetic map depicting the region of chromosome 2 that contains the MUSE9 locus, with markers used for mapping indicated. (B) Mutations identified within the mapping region of muse9 using Illumina sequencing.  (C) The gene structure of SYD, with the locations of the syd-4 and muse9 (syd-10) mutations indicated. Boxes and lines represent exons and introns, respectively. 38  (D) The conserved domain structure of the SYD protein, with the sites of the syd-4 and muse9 mutations denoted. Domains were identified using the NCBI Conserved Domain Database.  (E) Sequence comparison between wild-type SYD and muse9. A nucleotide substitution, indicated by the lower-case bolded ‘t’, results in an A2224V amino acid substitution.  The muse9 single mutant was obtained by backcrossing muse9 mos4 snc1 to Col-0 and selecting F2 lines that were homozygous for the muse9 mutation and wild-type at the MOS4 and SNC1 loci. Both the muse9 and syd-4 mutations result in slightly crinkled leaves and a small reduction in stature as compared to wild-type. The F1 progeny resulting from a cross of these two genotypes retain these characteristics (Figure 2.3A), indicating that muse9 failed to complement syd-4 and therefore that MUSE9 is SYD.  Figure 2.3. MUSE9 encodes SPLAYED (SYD), an ATP-dependent chromatin remodeler. (A) Complementation test between muse9 and syd-4. Morphology of soil-grown Col-0, snc1, mos4 snc1, muse9, syd-4, and an F1 plant from a cross between muse9 and syd-4. Photograph was taken 3 weeks post-germination. Scale bar indicates 1 cm. (B) Morphology of soil-grown Col-0, snc1, muse9, muse9 snc1, syd-4, and syd-4 snc1 plants. Photograph was taken 3 weeks post-germination. Scale bar indicates 1 cm.  39  As an additional method of verification, the snc1-enhancing effects of the two syd alleles were compared. The muse9 snc1 double mutant was isolated from the F2 progeny of the backcross described above, and the syd-4 mutant was crossed with snc1 to generate the syd-4 snc1 double mutant. Both double mutants show a dramatic reduction in size compared to either muse9 or syd-4 and snc1 (Figure 2.3B). Taken together, we conclude that the phenotypes associated with muse9 are a result of a mutation in SYD; therefore, we renamed muse9 as syd-10.  2.3.3 The syd-4 single mutant displays enhanced disease resistance As demonstrated above, syd-10 was found to enhance snc1-associated morphological and disease resistance phenotypes in the snc1 and mos4 snc1 genetic backgrounds. As growth of the syd-10 single mutant is slightly stunted (Figure 2.3A),  and fitness costs including diminished stature and reduced seed production are commonly associated with constitutive activation of NLR-mediated defense responses, it was hypothesized that the single mutant may show enhanced disease resistance independent of the presence of the snc1 mutation. This hypothesis was tested using a number of infection assays with virulent pathogens.  As noted above, snc1 displays enhanced resistance to the oomycete H.a. Noco2; however, resistance to this pathogen was found to be wild-type-like in both syd-4 and syd-10 (Figure 2.4A). snc1 also displays enhanced resistance to the virulent bacterial strain P.s.m. ES4326 (Zhang et al. 2003). When syd-10 and syd-4 plants were challenged with this pathogen the syd-10 single mutant was again found to display wild-type-like resistance, but enhanced resistance was consistently observed in the syd-4 single mutant (Figure 2.4B). We found that PR1 and PR2 are upregulated in both syd alleles (Figures 2.4C-D), although expression was enhanced to a greater degree in the syd-4 mutant. Consistent with a previous report which found 40  that expression of the defensin PDF1.2a, a marker of intact ET and JA signaling pathways, was reduced in the syd-2 mutant (Walley et al. 2008), we also observed lower PDF1.2a expression in the syd-4 and syd-10 mutants (Figure 2.4E). Since syd-4 contains an insertion in the conserved helicase domain of SYD while syd-10 carries a point mutation in the weakly conserved N terminal region of the protein (Figure 2.2D; Figure 2.5), it is possible that syd-10 is a weaker allele and therefore exhibits more subtle phenotypes.    41   Figure 2.4. The syd-10 single mutant does not display enhanced disease resistance. (A) Growth of H.a. Noco2 on indicated genotypes 7 d post-inoculation with 1x105 spores/mL. Values represent the average of 4 replicates of 5 plants each ± SD. (B) Growth of P.s.m. ES4326 on indicated genotypes 2 d post-infiltration. Values represent the average of 5 replicates ± SD. Significant difference between Col-0 and syd-4 indicated by *** (P-value < 0.001). (C-G) Real-time qRT-PCR analysis of (C) PR1, (D) PR2, (E) PDF1.2a, (F) SNC1, and (G) RPP4 expression in the indicated genotypes. Total RNA was extracted from seedlings grown for 12 d on MS media. Significant differences are indicated by asterisks (* P-value < 0.05, ** P-value < 0.01, *** P-value < 0.001). All experiments were repeated at least once with similar results.   42    Figure 2.5. Alignment of SYD proteins from a number of plant species. BLAST searches using the AtSYD amino acid sequence were performed, and the first hit for each of the indicated species was included in the alignment. A multiple sequence alignment was performed in BioEdit using ClustalW. The regions of the alignment containing the (A) syd-4 T-DNA insertion and (B) the syd-10 mutation are shown, with the sites of the mutations indicated by an asterisk (*).The numbers above the alignment correspond to the amino acid positions of the Arabidopsis thaliana sequence. At – Arabidopsis thaliana; Al – Arabidopsis lyrata; Os – Oryza sativa; Zm – Zea mays; Gm – Glycine max; Fv – Fragaria vesca; Pt – Populus trichocarpa; Rc – Ricinus communis; Tc – Theobroma cacao; Vv – Vitis vinifera; Sl – Solanum lycopersicum; Sm – Selaginella moelendorffii; Pp – Physcomitrella patens.  2.3.4 Mutations in SYD result in elevated transcription of SNC1 One mechanism to enhance disease resistance in plants is to increase steady-state levels of NLR proteins through transcriptional up-regulation. As SYD encodes an ATP-dependent chromatin 43  remodeler, it was hypothesized that the enhancement of snc1-like phenotypes associated with mutations in SYD may be a result of altered SYD function and subsequent changes in transcriptional activity at the SNC1 locus. Using real-time qRT-PCR, it was found that SNC1 expression is moderately but significantly elevated in both the syd-10 and syd-4 single mutants (Figure 2.4F). However, expression of RPP4, another NLR-encoding gene that resides within the same gene cluster as SNC1, was unaltered in the syd mutants (Figure 2.4G). These data suggest that SYD is responsible for maintaining proper transcript levels of SNC1 specifically. However, no obvious increase in SNC1 protein was observed in the syd single mutants (Figure 2.6). Without SYD function SNC1 transcription is up-regulated, which can be amplified in the snc1 mutant background and result in an enhancement of snc1-mediated autoimmunity.  Figure 2.6. SNC1 protein levels in the indicated genotypes. syd-5 is a T-DNA insertion allele (Salk_023209), and snc1-r1 is a null SNC1 allele which serves as a negative control. Two biological replicates of syd-4 are included. Signals detected using Ponceau staining served as internal loading controls.  2.4 Discussion Eukaryotic ATP-dependent chromatin remodeling complexes contain a DNA-dependent ATPase subunit which utilizes the energy derived from the hydrolysis of ATP to alter the positions of nucleosomes along the DNA strand (Clapier and Cairns, 2009). The resultant changes to 44  chromatin structure potentially modify the transcriptional activity at affected loci. One extensively studied ATPase in Arabidopsis is SYD, which belongs to the evolutionarily conserved SWI/SNF class of chromatin remodelers and was first identified as a regulator of reproductive development (Wagner and Meyerowitz 2002). Plant SWI/SNF chromatin remodeling complexes have been implicated in many biological processes in addition to development, including hormone signaling and RNA-mediated gene silencing (reviewed in Reyes 2014). In this study, we have determined a novel role for SYD in negatively regulating SNC1-mediated resistance. SYD was previously shown to be a regulator of JA- and ET-mediated stress signaling pathways and is required for resistance against Botrytis cinerea, a necrotrophic fungus with a broad host range (Walley et al. 2008). The same study reported that two mutant alleles of SYD conferred wild-type-like resistance to the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato DC3000, resistance against which is primarily mediated by SA. These results suggested that SYD is specifically involved in the regulation of disease resistance mediated by JA and ET signaling pathways, but not involved in SA-mediated immunity. Consistent with the previously published data, we found that the novel syd-10 allele also displays wild-type-like resistance to a different Pseudomonas syringae strain, P.s.m. ES4326 (Figure 2.4B). However, the syd-4 single mutant displays enhanced resistance to this pathogen. Differences in the immune phenotypes of syd-10 and syd-4 may be a result of the differing strengths of the mutations: syd-10 contains a point mutation in the weakly conserved N terminal region of SYD, while syd-4 carries a T-DNA insertion in the conserved helicase domain (Figure 2.2D). Additionally, the syd-10 allele confers enhanced resistance to the obligate biotrophic oomycete H.a. Noco2 in the mos4 snc1 genetic background (Figure 2.1C). The finding that SYD plays a role in mediating 45  resistance to biotrophic pathogens is not wholly unprecedented, as the SA-responsive defense marker gene PR1 was shown to be upregulated in syd-2, although none of the genes upstream in the SA signaling pathway were observed to have altered expression in the mutant (Walley et al. 2008). This supports our postulation that while SYD positively regulates JA- and ET-mediated defense against necrotrophs, it plays a role in the negative regulation of SA-mediated immunity.  From the phenotypic analysis of syd mutants, the role SYD plays in regulating SA-mediated defense responses appears to be quite subtle. This study has demonstrated that syd-10 enhances morphological and resistance phenotypes associated with snc1; however, the degree of the enhancement is not as strong as observed for other published muse mutants. The presence of the syd-10 mutation in the mos4 snc1 background only partially rescues the H.a. Noco2 resistance associated with snc1 (Figure 2.1C), and the immune phenotypes of the single mutant are almost indistinguishable from wild-type (Figure 2.4A-B), except for the enhanced resistance phenotype of the syd-4 single mutant. While SNC1 protein levels appear to be elevated in syd-10 mos4 snc1 as compared to mos4 snc1, SNC1 does not obviously accumulate in the syd single mutants (Figure 2.6). As SNC1 gene expression is only slightly increased in the syd mutants (Figure 2.4F), the consequent minute protein level change is likely below the detection limit of the western blot method. Given these results, it is unsurprising that syd alleles were not identified from any prior known screens for regulators of SA-mediated immunity. The sensitized genetic background used in the MUSE screen has enabled the identification of syd-10 and other novel components of immune signaling (Huang et al. 2013; Huang et al. 2014a; Huang et al. 2014b; Xu et al. 2015). One possible explanation as to why the defense phenotypes associated with the syd-10 mutation are only observable in the snc1 background is that in this background defense responses are constitutively activated; therefore, knocking out negative regulators of this 46  pathway results in a stronger, more quantifiable defense induction. In the wild-type genetic background, knocking out a minor negative immune regulator is insufficient to activate immune responses by itself; perhaps the threshold level of defense gene induction required to confer enhanced resistance cannot be reached. The mild effects of mutations in SYD upon SA-mediated signaling may also be partially explained by redundancy with its close homolog BRAHMA (BRM). These two ATPases have been demonstrated to act on both shared and unique target genes, and elevated expression of a number of SA-dependent defense response genes including PR1 has been observed in brm-101 mutants (Bezhani et al. 2007; Wu et al. 2012).  Other ATP-dependent chromatin remodeling complexes have been shown to repress SA-dependent defense gene expression. Mutations in subunits of the Arabidopsis SWR1 chromatin remodeling complex result in enhanced resistance to P.s.t. DC3000 and constitutive expression of genes associated with systemic acquired resistance (SAR), a long-lasting broad spectrum defense mechanism that protects against future infection and requires SA (March-Diaz et al. 2008). Such differential gene expression is caused by the loss of H2A.Z (March-Diaz et al. 2008), a histone variant important for regulating gene expression deposited by SWR1 complexes in plants, yeasts and mammals (Krogan et al. 2003; Kobor et al. 2004; Mizuguchi et al. 2004; Ruhl et al. 2006; Deal et al. 2007). In Arabidopsis, H2A.Z is enriched at genes responsive to environmental and developmental stimuli, such as genes involved in immune and temperature responses, and plays an essential role in controlling their expression (Coleman-Derr and Zilberman 2012; Kumar and Wigge 2010). Knocking out another chromatin-remodeling ATPase, DDM1, has been shown to release the suppression of SNC1 expression caused by the mos1 mutation, although expression of SNC1 in the ddm1 mutant is comparable to levels observed in 47  wild-type (Li et al. 2010). Taken together, these reports highlight the contribution of chromatin remodeling in defense gene regulation.  ATP-dependent chromatin remodelers are also known to affect DNA methylation, a type of epigenetic mark that can result in modified chromatin accessibility and gene transcription. As such, an examination of the DNA methylation status around the SNC1 locus was undertaken in syd plants. A slight decrease of DNA methylation in the asymmetric CHH (H = A, T or C) context was observed in syd at a transposon approximately 3 kb upstream of SNC1, as compared to wild type (Figure 2.7). To investigate if this is the cause of SNC1 transcriptional elevation in syd, we took advantage of mutants that exhibit reduced CHH methylation in this transposon (ddm1 and rdr2). No significant alteration of SNC1 expression was observed in either mutant (Figure 2.4F), indicating the suppression of SNC1 by SYD is unlikely to be mediated by DNA methylation at the SNC1 locus.   Figure 2.7. CHH methylation in wild type, syd-4, rdr2, and ddm1 plants around the SNC1 locus. DNA methylation was measured by bisulfite sequencing of genomic DNA from syd-4 and wild-type 3-week-old seedlings, and analyzed as previously described (Ibarra et al. 2012). rdr2-1 and ddm1-2 mutant data were obtained from Zemach et al. 2013. H = A, C, or T.  48   A graphic representation of the potential role of SYD in regulating SNC1-mediated immunity is illustrated in Figure 2.8.   Figure 2.8. SYD functions antagonistically with MOS1 and MOS9 to regulate SNC1 transcription. The chromatin remodeler SYD is a negative regulator of SNC1-mediated immunity. It may exert its regulatory effects by directly modifying chromatin at the SNC1 locus, thereby repressing SNC1 transcription (A). Alternatively, SYD may affect SNC1 transcription indirectly, by remodeling chromatin at a locus (or loci) elsewhere in the genome, thus affecting the expression of other regulators of SNC1 expression (B). SYD acts in opposition to previously reported MOS1, ATXR7 and MOS9, which function as positive regulators of endogenous SNC1 transcription.  SYD acts antagonistically to MOS1 and MOS9, and is required for negatively modulating transcription at the SNC1 locus. As part of the SWI/SNF complex, SYD may directly affect the SNC1 locus (Fig 2.8A). Alternatively, SYD may alter the chromatin at another locus (or loci), which indirectly results in the down-regulation of SNC1 transcription (Figure 2.8B). 49  Although MOS1 and MOS9 also affect RPP4 transcription (Li et al. 2010; Xia et al. 2013), SYD does not (Figure 2.4G), indicating that its effects on SNC1 are more specific.  In summary, we have shown that mutations in the ATPase-encoding gene SYD enhance the morphological and resistance phenotypes associated with the gain-of-function snc1 mutant and result in increased expression of SNC1. However, gaining comprehensive insight into the mechanism by which SYD regulates SNC1-mediated immunity requires further investigation.  2.5 Materials and methods  2.5.1 Plant growth conditions and mutant isolation Soil-grown plants were kept in climate-controlled growth rooms at 22ºC on a 16h light/8h dark cycle. Plate-grown plants were propagated on ½ Murashige and Skoog medium supplemented with 0.5% sucrose and 0.3% phytagel and grown under the above conditions. The MUSE screen was conducted using EMS as described previously (Huang et al. 2013). The syd-4 (Salk_149549) mutant was obtained from the Arabidopsis Biological Resource Center and genotyped by PCR using the following primers: 5’-TGAAGCTCTGACTTGCTCCTC-3’ and 5’-TCAAAGCAACAGACCATCGG-3’.  2.5.2 Expression analysis Approximately 0.1 g total plant tissue was collected from plate-grown 2-week-old seedlings. RNA was extracted using the Totally RNA Kit (Ambion, now Invitrogen), and Reverse Transcriptase M-MLV (Takara) was used to reverse transcribe 0.4 μg RNA. Primers used for 50  amplification of SNC1 and ACTIN7 were previously described (Zhang et al. 2003; Cheng et al. 2009).  2.5.3 Positional cloning Positional cloning of muse9 was performed using markers derived from insertion/deletion and single nucleotide polymorphisms between the Col-0 and Ler Arabidopsis ecotypes, identified using sequence information available from TAIR (Jander et al. 2002; http://www.arabidopsis.org). After narrowing down the location of the molecular lesion to between 10.8 MB and 12.4 MB, extracted DNA from muse9 mos4 snc1 was sequenced using the Illumina sequencing platform.  2.5.4 Pathogen assays Bacterial and oomycete infection assays were performed as previously described (Li et al. 2001). In brief, bacterial infections were conducting using a needleless syringe to infiltrate the abaxial leaf surfaces of 4-week-old soil-grown plants with P.s.m. ES4326 (OD600 = 0.001). Bacterial growth was quantified using leaf discs (area = 0.38cm2) collected on the day of infection (day 0) and 2 d later. Oomycete infections were conducted by spray-inoculating 2-week-old soil-grown seedlings with H.a. Noco2 (1x105 spores mL-1). Sporulation was quantified 7 d post-infection. Total aerial plant tissue was used in the assay. For each genotype, 5 replicates of 5 plants were each suspended in 1mL ddH20 and vortexed gently, and spores were counted using a hemocytometer. Spore counts were normalized to fresh weight (mg). 51  2.5.5 Genetic crosses The muse9 single mutant was generated by back-crossing muse9 mos4 snc1 with Col-0 containing the pPR2-GUS reporter gene. The F1 progeny were allowed to self-fertilize, and muse9 single mutants were identified among the F2 progeny by genotyping.    52  Chapter 3: A partial loss-of-function mutation in an Arabidopsis RNA polymerase III subunit leads to pleiotropic defects3  3.1 Summary  Plants employ five DNA-dependent RNA polymerases (Pols) in transcription. One of these polymerases, Pol III, has previously been reported to transcribe 5S rRNA, tRNAs, and a number of small RNAs. However, in-depth functional analysis is complicated by the fact that knockout mutations in Pol subunits are typically lethal. Here, we report the characterization of the first known viable Pol III subunit mutant, nrpc7-1. This mutant was originally isolated from a forward genetic screen designed to identify enhancers of the autoimmune mutant snc1, which contains a gain-of-function mutation in a nucleotide-binding leucine rich-repeat (NLR) immune receptor-encoding gene. The nrpc7-1 mutation occurs in an intron/exon splice site and results in intron retention in some NRPC7 transcripts. There is a global disruption in RNA equilibrium in nrpc7-1, exemplified by the altered expression of a number of RNA molecules, some of which are not reported to be transcribed by Pol III. There are developmental defects associated with the mutation, as homozygous mutants are dwarf, have stunted roots and siliques, and possess serrated leaves. These defects are possibly due to altered small RNA stability or activity. Additionally, the nrpc7-1 mutation confers an NLR-specific alternative splicing defect that correlates with enhanced disease resistance, highlighting the importance of alternative splicing in regulating NLR activity. Altogether, these results reveal novel roles for Pol III in maintaining                                                  3 A version of this chapter has been published. Kaeli C. M. Johnson, Yu Yu, Lei Gao, Ryan C. Eng, Geoffrey O. Wasteneys, Xuemei Chen and Xin Li. (2016) Journal of Experimental Botany 67(8):2219-30. 53  RNA homeostasis, adjusting the expression of a diverse suite of genes, and indirectly modulating gene splicing. Future analyses using the nrpc7-1 mutant will be instrumental in examining other unknown Pol III functions.  3.2 Introduction  Transcription under both static and dynamic conditions requires the action of evolutionarily conserved multi-subunit enzymes known as DNA-dependent RNA polymerases.  All eukaryotes possess three distinct RNA polymerases (Pols I, II, and III), each of which transcribes specific suites of genes (Cramer et al., 2008). Pol I transcribes 45S rRNA, which is the precursor to 5.8S, 18S and 25S rRNAs. Pol II transcribes mRNA as well as most small nuclear (sn)RNAs and micro (mi)RNAs. Pol III was previously thought to be primarily required for the transcription of “housekeeping” genes such as those encoding 5S rRNA and tRNAs. However, recent reports indicate that the Pol III transcriptome is more diverse than formerly assumed (Dieci et al., 2007). There are two additional plant-specific RNA polymerases, Pol IV and Pol V, which are required for the biogenesis and functional activity of small interfering (si)RNAs (Haag and Pikaard, 2011). As knockout mutations in the genes encoding the subunits of Pols I, II, and III are lethal, there is a dearth of functional analysis of plant Pols. While there are a number of published studies examining global transcriptomic changes in plants under various conditions (e.g. Nagano et al., 2012, Woo et al., 2012, Zhu et al., 2012), the literature to date has largely focussed on the roles played by Pol II-transcribed RNAs in regulating plants’ responses to stimuli. Stimulus-induced alteration of expression of protein-54  coding genes has been extensively documented. Numerous recent reports have highlighted the importance of miRNAs in regulating a broad spectrum of biological processes including development (Wu, 2013), flowering time (Spanudakis and Jackson, 2014), drought stress (Ding et al., 2013), metal toxicity (Gupta et al., 2014), immunity (Staiger et al., 2013), and phytohormone crosstalk (Curaba et al., 2014), among others. Furthermore, the biosynthesis, functional mechanisms, and degradation pathways of miRNAs have been well-studied (Rogers and Chen, 2013).  Comparatively little is known about Pol III-transcribed RNAs and how they aid plants in responding to intrinsic and extrinsic signals. An RNA molecule with significant sequence and structural similarity to 5S rRNA was found to regulate alternative splicing of certain pre-mRNAs in Arabidopsis (Hammond et al., 2009). Intriguingly, studies in a variety of eukaryotes indicate that Pol III-transcribed non-coding RNAs may play regulatory roles in addition to their housekeeping functions (Hu et al., 2012).  Among the various stimuli to which plants are subjected, biotic stress in the form of pathogenic infection requires that plants be able to respond rapidly and initiate signaling cascades specific to the type of pathogen being encountered. While plants possess physical barriers and broad spectrum resistance that is activated by conserved features of pathogenic microbes, many pathogens are able to inject infection-promoting effector molecules into the plant cell, thereby bypassing this layer of plant immunity (Bigeard et al., 2015). However, the plant genome contains a large number of genes encoding nucleotide-binding leucine rich-repeat proteins (NLRs; also referred to as Nod-like receptors due to their structural similarity to mammalian proteins of the same name), which either directly bind to pathogenic effectors or detect their activities within the plant cell in a highly specific manner (Li et al., 2015). Upon 55  recognition of its cognate effector, NLR activation results in rapidly induced and robust defense responses. Plant NLRs can be sorted into two classes based on their N-termini: some possess a Toll-Interleukin 1 receptor (TIR) domain and are thus termed TNLs, while others contain a coiled-coil (CC) domain and are referred to as CNLs.  NLR-mediated signaling must be tightly controlled under both resting and induced conditions, as improper signaling through this pathway may lead to either enhanced disease susceptibility or autoimmunity. However, the regulatory mechanisms underlying NLR-mediated signaling are only partially understood. A successful forward genetic suppressor screen previously conducted in our lab used the gain-of-function autoimmune TNL mutant snc1 (suppressor of npr1, constitutive 1; Li et al., 2001; Zhang et al., 2003) to search for positive regulators of immunity (Johnson et al., 2012). More recently, we have undertaken a forward genetic screen to identify negative regulators of NLR-mediated immunity.  Here, we report the characterization of nrpc7-1, a partial loss-of-function allele of the gene encoding the Arabidopsis ortholog of yeast Rpc25, a Pol III subunit. This mutant was isolated from our MUSE (MUTANT, snc1-ENHANCING) forward genetic screen conducted in the mos4 (modifier of snc1 4) snc1 double mutant background. A null mutation in NRPC7 is lethal, while a mutation in an intron/exon splice site junction gives rise to intronic retention in some NRPC7 transcripts, resulting in viable mutant plants. While the nrpc7-1 mos4 snc1 triple mutant displays enhanced resistance against the virulent oomycete pathogen Hyaloperonospora arabidopsidis Noco2, the nrpc7-1 single mutant exhibits wild type-level resistance. This correlates with the altered splicing of SNC1 observed in the triple mutant but not in the single mutant. Morphologically, the nrpc7-1 mutant is dwarf and has serrated leaves, short roots, and stunted siliques, although flowering time does not appear to be affected. The expression and 56  potentially activity of a number of RNAs are distorted in nrpc7-1, contributing to its developmental defects. In keeping with its known function, we observed that the NRPC7 protein localizes to the nucleus. This is the first reported viable Pol III subunit mutant in Arabidopsis.  3.3 Results  3.3.1 The isolation, characterization, and identification of the muse4/nrpc7-1 mutant The MUSE screen was designed to identify enhancers of the dwarf autoimmune mutant snc1 and has been described previously (Huang et al., 2013). To avoid potential lethality resulting from dramatically enhanced autoimmunity, the snc1 suppressor mos4 was included in the genetic background of the screen. Seeds from the wild type-like mos4 snc1 plants were mutagenized with ethyl methanesulfonate, and the M2 population was screened for plants displaying a reversion back to snc1-like morphology and resistance. A number of mutant lines were isolated, one of which (muse4) was selected for further characterization. When the triple mutant was backcrossed to mos4 snc1, all progeny appeared wild type-like, indicating that muse4 is a recessive mutation. As shown in Figure 3.1A, the muse4 mos4 snc1 plants exhibit dwarf, curled leaf morphology similar to that observed for snc1 plants. In addition, the muse4 mos4 snc1 plants have serrated and slightly chlorotic leaves. The muse4 mutation also re-establishes the constitutive expression of the defense marker PR (PATHOGENESIS-RELATED) genes observed in snc1 but absent in mos4 snc1. A pPR2-GUS reporter gene construct was used to visualize PR2 gene expression in seedlings, and GUS staining was much stronger in the triple mutant than in 57  mos4 snc1 (Figure 3.1B). Consistent with this observation, qPCR demonstrated that expression of PR1 and PR2 is elevated in the triple mutant (Figure 3.1C).   Figure 3.1. Characterization of the muse4 mos4 snc1 triple mutant. (A) Morphology of soil-grown plants of the indicated genotypes, photographed four weeks post-germination. Scale bar represents 1cm. (B) pPR2-GUS expression in seedlings of the indicated genotypes grown on MS media for 10 d. (C) PR1 and PR2 gene expression in the noted genotypes, as determined by qPCR. ACTIN7 expression serves as a loading control. (D) Growth of H.a. Noco2 on indicated genotypes 7 d post-inoculation with 1x105 spores/mL. Values represent the average of 4 replicates of 5 plants each ± SD. **: p-value ≤ 0.01; ***: p-value ≤ 0.001.  58  To examine whether the muse4 mutation alters resistance to the virulent oomycete strain Hyaloperonospora arabidopsidis Noco2, two-week-old triple mutant seedlings were spray-inoculated with this pathogen. The enhanced resistance observed in snc1 but lost in mos4 snc1 was found to be reconstituted in the triple mutant (Figure 3.1D). Together, these data indicate that muse4 restores all examined snc1-like phenotypes in the mos4 snc1 background. A positional cloning strategy was employed to determine the molecular lesion responsible for the observed phenotypes. The muse4 mos4 snc1 mutant, which was generated in the Col-0 ecotype, was crossed to Landsberg erecta (Ler). From the F2 population, 24 plants displaying the triple mutant morphology were selected for crude mapping, which identified a linkage to the top of chromosome 1. Several F2 plants heterozygous at the top of chromosome 1 (but homozygous for snc1 and mos4 to prevent interference by these loci) were used to generate a fine mapping population of approximately 500 plants. The mutation was narrowed down to between the markers T7A14 (1.4 MB) and F22O13 (2.75 MB). Genomic DNA was extracted from muse4 mos4 snc1 triple mutant plants and sequenced using the Illumina whole-genome sequencing platform. The sequencing results were compared with the Arabidopsis reference genome, and five genes in this region were found to contain mutations (Figure 3.2A). The mutations in three of these genes are located in introns and the mutation in one gene was found to be silent, therefore the mutation in the remaining gene (At1g06790) was selected as the most likely candidate for muse4. This gene encodes the Arabidopsis ortholog of the yeast Pol III subunit Rpc25, NRPC7 (NUCLEAR RNA POLYMERASE C, SUBUNIT 7; Ream et al., 2015), and the muse4 mutation is at the intron/exon junction just before the sixth exon (Figure 3.2B).  59   Figure 3.2. Map-based cloning of the muse4 locus on chromosome 1.  (A) Point mutations identified within the mapping region from Illumina sequencing of the muse4-1 snc1 mos4 triple mutant. Two independent mutations in introns were identified in AT1G05570.  (B) The two alternatively spliced variants of MUSE4 and the position of the molecular lesion in muse4-1 (nrpc7-1) and muse4-2 (nrpc7-2). Boxes and lines represent exons and introns, respectively.  3.3.2 The mutation at an intron/exon junction of NRPC7 results in intron retention and is responsible for the muse4 phenotypes To verify that the mutation in NRPC7 is responsible for the muse4 phenotypes, a full-length wild type copy of the gene driven by its native promoter and fused to the GFP-encoding gene at its 3’ end was transformed into the single mutant, which was generated by backcrossing the triple mutant to Col-0 and selecting plants homozygous for wild type SNC1 and MOS4 that retained the serrated leaf phenotype and dwarf size. Eight independent T2 lines displayed wild type 60  morphology, and one representative line can be seen in Figure 3.3A. These data suggest that NRPC7 can fully complement the muse4 phenotypes, and therefore that MUSE4 is indeed NRPC7.   Figure 3.3. MUSE4 is NRPC7. (A) MUSE4 tagged with GFP and expressed under the control of its native promoter is able to complement the muse4 single mutant defects. Plants were grown on soil for three weeks. (B) The size of the MUSE4 transcript in wild type and muse4 was examined using cDNA reverse-transcribed from total RNA. (C) The larger muse4 band in (B) was excised, purified, and sequenced, and found to retain the intron preceding the intron/exon splice site mutation in muse4.  61  We hypothesized that the muse4 mutation in the intron/exon junction of NRPC7 results in retention of the preceding intron. To test this, we designed primers flanking the intron of interest and amplified cDNA from wild type and muse4. A strong band of the expected size (465 bp) was observed in wild type while in muse4 two bands were observed, one of the expected size and one slightly larger (Figure 3.3B). The larger band was excised from the gel and the PCR product was purified and sequenced. As predicted, sequencing revealed that the larger band corresponded to a transcript in which the intron preceding the muse4 mutation had been retained (Figure 3.3C). Despite strong sequence similarity between NRPC7 and known Rpc25 proteins in other species (Figure 3.4), the NRPC7 gene failed to complement a temperature sensitive rpc25 yeast knockout line (Figure 3.5), suggesting divergence between the plant and yeast NRPC7.  Figure 3.4. Sequence alignment of RPC25 from a broad range of species, based on BLAST analysis. 62   Figure 3.5. Yeast complementation with NRPC7. NRPC7 CDS was introduced into two independent temperature-sensitive rpc25 knockout yeast lines. WT: yeast rpc25 strain with no transgene; EV: rpc25 transformed with an empty vector; C: rpc25 transformed with the NRPC7-containing construct.  It is expected that a knockout mutation in NRPC7 would be embryo lethal, as a previous study showed that loss-of-function mutations in RNA polymerase subunits are not transmitted maternally (Onodera et al. 2008). Indeed, when we let the heterozygous nrpc7 T-DNA insertion line CS1001213 self-fertilize and then planted the progeny, we identified 23 wild type plants lacking the insertion, 46 heterozygotes, and 0 plants that were homozygous for the insertion, matching the expected 1:2:0 (wild type:heterozygote:homozygote) ratio for a lethal mutation. We also performed reciprocal crosses between this heterozygous T-DNA insertion line and muse4 and found that none of the F1 progeny contained the T-DNA insertion, indicating that the T-DNA/muse4 heterozygotes are not viable. These results, combined with the data in Figure 3.3B showing that muse4 still produces some properly spliced transcripts without intron retention, as well as the fact that muse4 is a recessive mutation, suggest that muse4 is a partial loss-of-63  function allele of NRPC7. Therefore, we renamed muse4 as nrpc7-1 and the T-DNA allele as nrpc7-2.  3.3.3 Splicing of SNC1 is altered in the nrpc7-1 mos4 snc1 background In yeast, Rpc25 is required for Pol III transcription initiation (Zaros and Thuriaux, 2005). To assess whether Pol III function is affected by the nrpc7-1 mutation, we used real-time qPCR to determine whether expression of U6, a snRNA component of the spliceosome that is known to be transcribed by Pol III (Waibel and Filipowicz, 1990), is different in nrpc7-1 than in wild type. Relative to the expression levels of the Pol II-transcribed “housekeeping” gene UBQ5, U6 expression is significantly lower in nrpc7-1 (Figure 3.6A). To examine whether the nrpc7-1 mutation has a general effect on spliceosomal snRNA biosynthesis, the accumulations of Pol II-transcribed U1 and U2 snRNAs were also examined.  While U1 accumulation is wild type-like, U2 expression is significantly reduced in nrpc7-1. This is likely due to an indirect effect of altered Pol III function on Pol II-transcribed genes.  The reduced expression of the spliceosome components U6 and U2 lead us to hypothesize that pre-mRNA splicing might be affected by the nrpc7-1 mutation. Specifically, as nrpc7-1 was isolated in our screen for snc1 enhancers, we hypothesized that the mutation may affect the excision of introns from the SNC1 pre-mRNA transcript. The alternative splicing of a number of plant NLR-encoding genes, including SNC1, is known to affect their function in plant immunity (Yi and Richards, 2007; Xu et al., 2011). For SNC1, the second and third introns may be either retained or removed; therefore we used primers spanning these two introns to amplify the SNC1 transcript variants. A dramatic accumulation of the largest transcript variant (with both introns retained) was observed in nrpc7-1 mos4 snc1, although the SNC1 splicing pattern in the 64  nrpc7-1 single mutant was indistinguishable from that observed in wild type (Figure 3.6B; Figure 3.7C).   Figure 3.6. Splicing defects in nrpc7-1. (A) Quantitative real-time qPCR was used to determine the expression of Pol II-transcribed U1 and U2 snRNAs, as well as Pol III-transcribed U6, relative to UBQ5. Bars represent the averages of three technical replicates of two biological replicates ± SD. **: p-value ≤ 0.01. (B) An analysis of SNC1, RPS4, SR30, and PAD4 splicing patterns in the indicated genotypes was performed using RT-PCR. Transcripts were amplified using 40 cycles. Numbers indicate transcript variants from largest to smallest. Schematic diagrams of the expected splicing events are shown to the right, with horizontal lines representing introns, black boxes representing exons, and white boxes representing alternatively retained exons that result in a premature stop codon. (C) Quantification of the alternative transcript variants in (B) across genotypes. Band intensities were quantified using ImageJ. 65   Figure 3.7. SNC1 gene and protein expression in nrpc7.  (A) Real-time qPCR analysis of SNC1 expression in the indicated genotypes. Total RNA was extracted from seedlings grown for 12d on MS media. (B) Western blot analysis of SNC1 levels. Protein was extracted from seedlings grown on MS media for 12d. (C) The original SNC1 splicing pattern gel image. An altered version of this image was used in Figure 4.6B.  Alternative splicing defects were also observed in nrpc7-1 mos4 snc1 for RPS4 (RESISTANT TO PSEUDOMONAS SYRINGAE 4), another NLR-encoding gene, as well as for 66  SR30, which encodes a serine/arginine-rich RNA-binding protein and is known to be alternatively spliced (Figure 3.6B). The relative proportions of the transcript variants in the genotypes examined are shown in Figure 3.6C. These data reveal significant alternative splicing defects caused by the Pol III subunit mutation. To determine whether this splicing defect occurs at the level of basal splicing, transcripts of a gene that is not alternatively spliced (PAD4; PHYTOALEXIN-DEFICIENT 4) were also examined. No difference from the wild type splicing pattern was detected (Figure 3.6B). Both snc1 and nprc7-1 mos4 snc1 accumulated higher levels of PAD4 compared to wild type, which is consistent with previous reports that PAD4 is a defense-induced gene (Glazebrook 2001), whose expression is expected to be upregulated in autoimmune mutants. NLR-mediated signaling is often regulated by modulating transcription and/or translation of NLRs. As such, we examined whether SNC1 expression and protein accumulation are affected by the nrpc7-1 mutation. SNC1 expression was found to be slightly reduced in nrpc7-1 as compared to wild type, while SNC1 protein levels were wild type-like (Figure 3.7). Similarly, the accumulation of SNC1 in nrpc7-1 mos4 snc1 was not dramatically higher than that observed in mos4 snc1. Taken together, these data indicate that SNC1 alternative splicing, but not overall gene expression or translation, is affected by the nrpc7-1 mutation.   3.3.4 The nrpc7-1 single mutant does not have altered immune responses Since alterations in the splicing of SNC1 were observed in the nrpc7-1 mos4 snc1 triple mutant but not the nrpc7-1 single mutant, we predicted that nrpc7-1 may not exhibit the enhanced disease resistance observed in the triple mutant (Figure 3.1D). We challenged nrpc7-1 with the oomycete pathogen H.a. Noco2 and the bacterial pathogen Pseudomonas syringae pv. 67  maculicola ES4326, and found no statistically significant difference in response from that observed in wild type plants (Figure 3.8). These data support our hypothesis that the retention of introns in the SNC1 transcript in the nrpc7-1 mos4 snc1 confers enhanced disease resistance, and is the reason why this mutation was isolated from our snc1 enhancer screen.   Figure 3.8. Immune characterization of nrpc7-1 single mutant plants. (A) Growth of H.a. Noco2 on indicated genotypes 7 d post-inoculation with 1x105 spores/mL inoculum. Values represent the average of 4 replicates of 5 plants each ± SD.  (B) Growth of P.s.m. ES4326 on indicated genotypes 2 d post-infiltration. Values represent the average of 5 replicates ± SD.    3.3.5 nrpc7-1 has global defects in RNA levels  Pol III transcribes tRNA, 5S rRNA, and assorted other non-coding RNAs. To further explore how Pol III function is affected by the nrpc7-1 mutation, we examined the expression of a variety of RNAs by qPCR (Figure 3.9A). Relative to UBQ5, 5S rRNA and three representative tRNAs (coding for Gln, Gly, and Leu, respectively) showed significantly reduced accumulation in nrpc7-1 compared to wild type.  68   Figure 3.9. Global RNA defects in nrpc7-1. (A) Expression of several representative Pol III-transcribed RNAs was examined in Col-0 and nrpc7-1 using qPCR. Bars represent the averages of three technical replicates of two biological repeats ± SD.  (B) The proportions of rRNAs in the noted genotypes were compared by running total RNA extracted from seedlings grown on MS media for 12d on a 2% agarose gel. Band intensities were quantified using ImageJ, and the intensities of chloroplast rRNA (16S and 23S) relative to Pol I-transcribed rRNA (25S) were compared between genotypes for three biological replicates ± SD.  (C) The accumulation of CUC1, CUC2, PHB, and REV transcripts was examined using quantitative real-time qPCR. Bars represent the averages of three technical replicates of two biological replicates ± SD. *: p-value ≤ 0.05; **: p-value ≤ 0.01; ***: p-value ≤ 0.001; ****: p-value ≤ 0.0001.  69  In addition, when total RNA was run on a 2% agarose gel, altered relative proportions of the various rRNAs were consistently observable in association with the nrpc7-1 allele (Figure 3.9B). Relative to Pol I-transcribed 25S rRNA, there appears to be a lower abundance of chloroplast 16S and 23S rRNA associated with the nrpc7-1 allele, although as chloroplast numbers were not examined in the mutant we cannot rule out the possibility that nrpc7-1 affects chloroplast abundance. These data suggest that in addition to Pol III transcribed genes, the nrpc7-1 mutation also affects abundance of other RNAs, likely through indirect mechanisms. Small RNA libraries were then prepared from BG2 plants (Col-0 with the pPR2-GUS reporter gene construct that is present in the nrpc7-1 background) and two independently isolated nrpc7-1 single mutant lines. Analysis of these small RNA libraries indicated that a number of miRNAs are differentially expressed in the mutant. Those miRNAs that exhibited a two-fold or greater change in expression are shown in Figure 3.10.   Figure 3.10. RNA defects in nrpc7-1.  (A) The expression of a number of miRNAs, quantified as reads per million, based on an analysis of small RNA libraries. Only miRNAs that had a fold change ≥ 2 between wild type and nrpc7-1 and an FDR < 0.05 are included. Bars are representative of two biological replicates.  70  (B) Northern blot analysis of a select number of miRNAs that small RNA library analysis indicated were differentially expressed in nrpc7-1. Numbers indicate the average and standard deviation of two technical replicates of two biological replicates.   To validate these results, three representative miRNAs were selected for northern blot analysis. Although the data from the small RNA libraries indicated that expression of both miR159 and miR166 is reduced in nrpc7-1 while miR398 expression is increased, no significant alterations in the levels of these miRNAs were consistently observed via northern blotting (Figure 3.10B), suggesting that any differences that exist between the mutant and wild type are too subtle to be detected by this method. The general disruption in RNA equilibrium combined with the striking serrated leaf phenotype and dwarf morphology of the nrpc7-1 mutant lead us to hypothesize that the expression of (i) the CUC (CUP-SHAPED COTYLEDONS) genes, which are targeted by miR164 (Mallory et al., 2004), and (ii) the HD-ZIP (HOMEODOMAIN-LEUCINE ZIPPER) genes, which are targeted by miR165/166 (Rhoades et al., 2002), might be altered in the mutant. Expression of a CUC2 transcript resistant to miR164 cleavage was previously shown to result in enhanced leaf serration; the same morphological phenotype was observed in plants containing loss-of-function mutations in the Pol II-transcribed MIR164 (Nikovics et al., 2006). Overexpression of either miR165 or miR166 results in reduced expression of the HD-ZIP genes, which corresponds with dwarf morphology and altered rosette leaf morphology that is similar to that observed in nrpc7-1 (Jung et al. 2007).   Real-time qPCR was used to determine that CUC1 and CUC2 accumulation is elevated in nrpc7-1 (Figure 3.9C), although no alteration in miR164 levels were observed in nrpc7-1 based on our small RNA library data. Expression of the HD-ZIP genes PHB (PHABULOSA) and REV 71  (REVULOTA) was found to be decreased in nrpc7-1 (Figure 3.9C). No changes in miR165 levels were observed in nrpc7-1, and although miR166 expression was elevated in the mutant according to the small RNA library sequencing data (Figure 3.10A) no detectable change in miR166 levels was consistently measurable by northern blotting (Figure 3.10B). The altered expressions of CUC1, CUC2, PHB, and REV in the absence of detectable changes in miR164, miR165, and miR166 abundances suggests that in nrpc7-1 the activity of a number of small RNAs may be affected; alternatively, the nrpc7-1 mutation may indirectly affect Pol II-mediated transcription of certain genes, including CUC1, CUC2, PHB, and REV, although the mechanism behind this specificity is unclear. Many of the RNAs that seem to be differentially expressed in nrpc7-1 are not transcribed by Pol III (Figure 3.9), suggesting that this mutation results in a disruption of the global RNA equilibrium and homeostasis.  3.3.6 NRPC7 localizes to the nucleus As part of the Pol III complex, NRPC7 is predicted to localize to the nucleus. To examine its localization, we used the complementing nrpc7-1 lines containing the transgene with NRPC7 fused to GFP under the control of the native promoter, described above. We analyzed cotyledon and root tissue using confocal microscopy, and GFP fluorescence was visible throughout the nucleus as it co-localized with nuclei stained with propidium iodide (Figure 3.11). Additionally, there appeared to be intense fluorescent foci within the nucleus and along the plasma membrane. 72   Figure 3.11. Subcellular localization of NRPC7-GFP. NRPC7-GFP as observed by confocal microscopy in cells from the cotyledon (A) and root (B) from Arabidopsis seedlings grown for 12d on MS media. Propidium iodide (red) was used to stain the cell wall and nuclei. Scale bars represent 20 µm. n = nucleus.  3.3.7 nrpc7-1 has pleiotropic developmental defects The roles various small RNAs play in the regulation of plant development have been well studied. As nrpc7-1 has large impacts on small RNA levels and, potentially, RNA activities, we examined the developmental phenotypes of the mutant. As described earlier, nrpc7-1 has serrated leaves (Figure 3.3A; Figure 3.12A), and its growth is stunted (Figure 3.12B). When grown on half-strength MS media, nrpc7-1 plants also have significantly shorter roots than wild type plants (Figure 3.12C). The siliques of nrpc7-1 were consistently found to be smaller (Figure 3.12D). Flowering time was measured using several different assays, but a significant difference between nrpc7-1 and wild type was only observed when measuring the number of days until the primary stalk reached 6 cm (Figure 3.12E), which is likely a reflection of the restricted growth of the mutant rather than an actual delay in flowering time. While the number and arrangement of the floral organs are wild type-like, the texture of the sepals is bumpy and irregular (Figure 73  3.12F). These results show that the nrpc7-1 mutation is associated with a number of pleiotropic developmental defects.   Figure 3.12. Developmental defects of the nrpc7-1 mutant. (A) Rosette leaf morphology from wild type and nrpc7-1 plants at bolting. Bar indicates 1 cm. (B) Morphology of soil-grown plants eight weeks post-germination. Scale bar represents 1cm. (C) Comparison of root length in cm between wild type and nrpc7-1. Seedlings were grown vertically on ½ MS for 14d. Bars represent five replicates ± SD. ****: p-value ≤ 0.0001. (D) Comparison of silique length between wild type and nrpc7-1. Siliques were harvested from mid-level of the primary stem for each plant. Bars represent three replicates of five siliques per plant ± SD. ***: p-value ≤ 0.001. (E) Four different approaches were used to investigate flowering time in wild type and nrpc7-1 plants under both short day and long day conditions. (F) Morphology of wild type and nrpc7-1 flowers. Scale bar indicates 0.5cm.  74  3.4 Discussion We demonstrated that Pol III function is altered by the partial loss-of-function mutation nrpc7-1 by showing that the expression of Pol III-transcribed U6 snRNA, 5S rRNA, and a number of tRNAs are reduced in the mutant (Figure 3.6A; Figure 3.9A) Pol II-transcribed U2 snRNA, but not U1 snRNA, also had reduced expression in nrpc7-1 (Figure 3.6A), indicating that the transcriptional defects in the mutant extend to genes not directly transcribed by Pol III. The decreased accumulation of U6 and U2 snRNA lead us to hypothesize that spliceosome functionality is impaired in nrpc7-1 and that alternative splicing of SNC1 is consequently affected, thereby explaining why this mutation was isolated from a screen for enhancers of the autoimmune mutant snc1. Indeed, SNC1 splicing is defective in the nrpc7-1 mos4 snc1 triple mutant background, in that there is a dramatic accretion of a transcript variant that retains both the second and third introns (Figure 3.6B-C). A similar pattern was observed for the NLR-encoding gene RPS4. The second intron of SNC1 contains an in-frame premature stop codon, thus retention of this intron should yield a truncated version of the protein. Previous reports have shown that an accumulation of the N termini of TNL proteins is sufficient to activate cell death and immunity (Weaver et al., 2006; Swiderski et al., 2009). This finding, combined with our data showing that transcription and translation of SNC1 are not enhanced by the nrpc7-1 mutation (Figure 3.7A-B), suggests that the modification in SNC1 splicing could be the primary cause of the snc1 enhancing effects of the nrpc7-1 mutation in the mos4 snc1 background (Figure 3.1).  It is notable that the nrpc7-1 single mutant does not differ from wild type in either alternative splicing (Figure 3.6B-C) or disease resistance (Figure 3.8). This suggests that the mos4 mutation is required in the genetic background for the nrpc7-1-associated splicing defects 75  to become obvious. MOS4 is an integral component of the evolutionarily conserved MOS4-associated complex (MAC) that functions together with the spliceosome to regulate pre-mRNA splicing (Johnson et al., 2011). Mutations in mos4 and other MAC components have previously been shown to affect the alternative splicing of both SNC1 and RPS4 (Xu et al., 2012), and are associated with a suppression of SNC1-dependent immune signaling (Palma et al., 2010). In this study we demonstrated that there is an increased accumulation of the intron-retaining SNC1 transcripts in mos4 snc1 compared with snc1 (Figure 3.6C). However, this accumulation is radically enhanced in the nrpc7-1 triple mutant. One possible explanation for these results is that the mos4 mutation and, to a lesser extent the nrpc7-1 mutation, individually disrupt splicing efficiency, reducing the pool of the functional full-length SNC1 transcript variant with both introns excised but not increasing the production of alternative variants beyond the threshold required for immune activation. However, when these two mutations are combined in the snc1 background, spliceosome activity is markedly disturbed and the accumulation of intron-retaining SNC1 transcript variants is sufficiently high to yield enough truncated SNC1 to activate defense responses.  In addition to the splicing defects observed in nrpc7-1, the accumulations of rRNAs and tRNAs appear to be considerably distorted (Figure 3.9A-B). Ribosomal protein gene dosage was recently found to have an effect on embryonic stem cell differentiation in mice (Fortier et al., 2015), indicating that alterations in the abundance of ribosome components can dramatically alter developmental progression. Homozygous nrpc7-1 plants exhibit certain phenotypes that may be associated with impaired stem cell differentiation including short roots (Figure 3.12C) and delayed emergence of the first true leaves. This suggests that the sensitivity of ribosome function to changes in its subunit levels, as well as its role in regulating stem cell differentiation, 76  may be conserved in plants, although the data in support of this is preliminary and additional experiments are required to fully explore this hypothesis.  We also detected a reduction in the accumulation of the chloroplast 16S and 23S rRNAs relative to Pol I-transcribed 25S rRNA (Figure 3.9B). A similar rRNA abundance pattern was recently reported for atybeY-1, a mutant allele of an endoribonuclease required for chloroplast rRNA processing and development that also exhibits pale green leaves and delayed development (Liu et al., 2015). Although the mechanism by which alterations in a Pol III subunit result in changes to the transcriptional regulation of Pol I-transcribed genes and the chloroplast genome is unclear, the light green colour of the nrpc7-1 single mutant (Figure 3.3A) further suggests that this mutation may be associated with impaired chloroplast function. The serrated leaf phenotype observed in nrpc7-1 is likely linked to its elevated expression of CUC1 and CUC2 (Figure 3.9C), which could be a result of reduced miR164 activity in the mutant background or an indirect effect of the nrpc7-1 mutation on Pol II function. There may also be a link between the nrpc7-1 mutant morphology and the decreased expression of the HD-ZIP genes (Figure 3.9C). Other studies have demonstrated tentative links between the transcriptional activities of Pol II and Pol III. One study identified areas of the genome where protein-coding genes on one DNA strand overlapped with tRNA-encoding genes on the opposite strand, and that their rates of transcription by Pol II and Pol III, respectively, were negatively correlated (Lukoszek et al., 2013). Another study found that human RPPH1 is transcribed by both Pol II and Pol III, and identified a number of transcriptional activators that associate with both Pols (Faresse et al., 2012). Our data show that disturbances in Pol III function affects the expression of non-Pol III-transcribed RNAs, indicating that the role of Arabidopsis Pol III in transcriptional regulation is more complex than previously assumed. 77  There are twelve core subunits of Arabidopsis Pol III, each of which has a homolog or is itself also a component in Pols I, II, IV, and V (Haag and Pikaard, 2011; Ream et al., 2015). There are also subunits specific to individual Pols. NRPC7 encodes a core Pol III subunit with homologs in each of the other Pols, and shares significant sequence similarity with Rpc25 proteins from other model organisms (Figure 3.4). However, Arabidopsis NRPC7 failed to complement a temperature-sensitive rpc25 yeast knockout line (Figure 3.5), suggesting that the functional conservation of this protein by itself between yeast and plants is limited. This is not entirely unprecedented. Rpc25 is known to form a dimer with Rpc17 within the Pol III complex (Siaut et al., 2003). The protein-protein interaction surface of NRPC7 may be sufficiently evolutionarily divergent so as to prohibit it from dimerizing with yeast Rpc17. Although the function of the protein complex is conserved, an individual component of the complex may still be divergent enough that it fails to complement a knockout of its ortholog in a distant organism.  In summary, we have demonstrated that a perturbation in Pol III function results in modified gene splicing as well as alterations in the abundances and potentially activities of a number of RNA molecules. These effects extend to several RNAs reported to be transcribed by other polymerases, revealing a novel role for Pol III in modulating the expression of a larger complement of genes than previously described. Moving forward, the partial loss-of-function nrpc7-1 mutant provides a unique tool for performing other functional analyses of Pol III.    78  3.5 Materials and methods  3.5.1 Plant growth conditions and mutant isolation Plants were grown either on soil or on half-strength Murashige and Skoog (MS) media supplemented with 1% sucrose and 0.3% phytagel. All plants were grown under long day conditions (16 h light/8 h dark) at 22ºC in climate-controlled chambers. The muse4 mutant was isolated from the MUSE screen, described previously (Huang et al., 2013).   3.5.2 Total RNA extraction and analysis Approximately 0.1 g tissue was collected from 2-week-old seedlings grown on ½ MS, and the Totally RNA Kit (Ambion, now Invitrogen) was used to extract total RNA. For the comparison of rRNA levels, total RNA was run on a 2% agarose gel. To reverse transcribe 0.4 μg RNA to cDNA, the Reverse Transcriptase M-MLV (Takara) was used after treating the RNA with DNaseI (Promega). The sequences of primers used were: 4F 5’-AATCTCCCTCTCGAAGATGC-3’ and 4R 5’-AAAGGCTTTGCGTCCTCTGC-3’ for MUSE4/NRPC7; U1F 5’-TACCTGGACGGGGTCAAC-3’ and U1R 5’-CCCTCTGCCACAAATAATGAC-3’ for U1; U2F 5’-TCGGCCCACACGATATTAAC-3’ and U2R 5’-GCAGTAGTGCAACGCATAGG-3’ for U2; 5SF 5’-GGATGCGATCATACCAGC-3’ and 5SR 5’-GAGGGATGCAACACGAGG-3’ for 5S rRNA; 7SLF 5’-CAAATCAAGTGGTTCAACCC-3’ and 7SLR 5’-CTTCGACGTTATCATCTGCG-3’ for 7SL RNA; GlnF 5’-GGTTCTATGGTGTAGTGGTTAGC-3’ and GlnR 5’-TACCGGGAGTCGAACCCAG-3’ for tRNA-Gln; GlyF 5’-GCACCAGTGGTCTAGTGGTA-3’ and GlyR 5’-TGCACCAGCCGGGAATCGAA-3’ for tRNA-Gly; and LeuF 5’-79  TGTCAGAAGTGGGGTTTGAACC-3’ and LeuR 5’-TCAGGATGGCCGAGTGGTCTAA-3’ for tRNA-Leu. Primers used for amplification of SNC1, RPS4, SR30, PAD4, and ACTIN7 were previously described (Zhang et al., 2003; Cheng et al., 2009; Xu et al., 2012).  3.5.3 Infection assays H.a. Noco2 infection was performed by spraying 2-week-old soil-grown seedlings with a spore suspension with a concentration of 105 spores per mL of water. Inoculated seedlings were grown for 7 d at 18ºC in a growth chamber with ~80% humidity and a 12 h light/12 h dark cycle. Sporulation was then quantified using a hemocytometer to count the number of spores from five plants shaken in 1 mL of water. Five replicates were performed for each of three independent trials. P.s.m. ES4326 infection was performed by infiltrating the abaxial leaf surface of 4-week-old soil-grown seedlings with bacteria suspended in 10 mM MgCl2 (OD600=0.0005). Leaf punches were collected at day 0 and day 3, and serial dilutions were performed and plated on LB media. Plates were incubated at 28ºC for 24 h before colony forming units were measured.   3.5.4 Positional cloning and Illumina whole-genome sequencing Positional cloning of muse4 was performed by crossing the muse4 mos4 snc1 triple mutant (generated in the Col-0 ecotype) with wild type Landsberg erecta. 24 F2 plants homozygous for all three mutations were used for crude mapping, and approximately 500 F3 plants homozygous for mos4 and snc1 and heterozygous for muse4 were used for fine mapping. The markers used in mapping were derived from insertion/deletion polymorphisms between the Col-0 and Ler Arabidopsis ecotypes (Jander et al., 2002; http://www.arabidopsis.org). After determining that the mutation must be located on the top of chromosome 1 between 1.4 MB and 2.75 MB, 80  extracted genomic DNA from muse4 mos4 snc1 was sequenced using the Illumina sequencing platform.  3.5.5 Preparation of transgenic plants and confocal microscopy Full length At1g06790 genomic DNA, including 766 bp upstream of the start codon, was amplified via PCR, cloned into the pCambia1305 vector, and transformed into muse4 mos4 snc1 using the floral dip method (Clough and Bent, 1998). The full length genomic fragment was also cloned into a pCambia1305 vector containing a GFP tag. Transgenic plants were selected for on ½ MS plates containing 50 mg/mL hygromycin. Confocal images of wild type (negative control), 35S::X-GFP (positive control), and NRPC7-GFP transgenic seedlings were obtained using a Perkin Elmer Ultraview VoX Spinning Disc Confocal system (Perkin-Elmer) mounted on a Leica DM16000 B inverted microscope and equipped with a Hamamatsu 9100-02 electron multiplier CCD camera (Hamamatsu). An argon 488 nm laser line with a complementary (522/36) emission band-pass filter to detect GFP or a 561 nm laser with a complementary (595/50) emission band-pass filter to detect propidium iodide was used. Images were acquired with a 63x (water) objective lens. To stain the nuclei and the cell wall, seedlings were incubated in a 10 µg/mL solution of propidium iodide (Calbiochem) for 1 min, rinsed with water, and mounted on a slide and coverslip prior to imaging.  3.5.6 Yeast complementation Full length MUSE4 cDNA was cloned into the yeast expression vector p425-GPD with primers 5’-CGCggatccATGTTTTATCTTAGCGAGC-3’and 5’- ACGCgtcgacTCACTCTTCTTGATCAACC-3’, using BamHI and SalI digestion sites. MUSE4 81  and empty vector control plasmids were introduced into the yeast rpc25-ts strain using a standard polyethylene glycol/lithium acetate yeast transformation protocol (http://labs.fhcrc.org/gottschling/Yeast%20Protocols/ytrans.html). Yeast transformants were grown overnight, serially diluted, and plated onto SD-Leu plates grown under either 28ºC or 37ºC to assay for growth.  3.5.7 Small RNA library construction and sequencing Small RNAs within the size range of 15nt to 40nt were fractionated from total RNAs by 15% polyacrylamide gel electrophoresis. These small RNAs were then ligated with the 3' and 5' adapters sequentially using the Small RNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions. A reverse transcription reaction followed by a low cycle PCR was performed to obtain final products for deep sequencing. The wild type and muse4 libraries were barcoded and sequenced in one channel on an Illumina Hiseq2000.   3.5.8 Analysis of small RNA high throughput sequencing data PERL scripts were used to process small RNA raw reads as per Lertpanyasampatha et al. (2012).  To summarize, reads were passed through Illumina’s quality control filter before being sorted into bins based on their barcodes and having their adaptor sequences removed. SOAP2 was used to map reads within the size range of 20–24 nt to the Tair10 Arabidopsis genome (Li et al., 2009). Differential small RNA regions were identified as previously described (Dinh et al., 2014). For analysis of differentially expressed miRNAs, all known Arabidopsis miRNAs were downloaded from miRBase (Release 20 from www.mirbase.org; Griffiths-Jones et al., 2008). PERL scripts were used to determine the expression level of known miRNAs in the small RNA 82  libraries and then normalize these counts to RPM (reads per million). miRNAs with < 10 RPMs in both nrpc7-1 and wild type libraries were removed. The differentially expressed miRNAs were identified by comparing expression in the nrpc7-1 library with wild type. The Audic-Claverie method was used to calculate P-values (Audic and Claverie, 1997), which were subsequently adjusted as described by Benjamini and Hochberg (1995) to determine the false discovery rate (FDR). To qualify as a differentially expressed miRNA, both a fold change ≥ 2 between wild type and nrpc7-1 and an FDR < 0.05 were necessary.     83  Chapter 4: The putative kinase substrate MUSE7 negatively regulates the accumulation of SNC1  4.1 Summary  The strict regulation of immune signaling in plants is required in order to enable rapid response to pathogen attack as well as to prevent spurious activation of defense responses that may be associated with fitness costs. However, these regulatory mechanisms are only partially understood. To identify novel negative regulators of plant immunity, a forward genetic screen was designed to look for enhancers of the dwarf autoimmune snc1 (suppressor of npr1, constitutive 1) mutant. The screen was conducted using wild-type-like mos4 (modifier of snc1, 4) snc1 plants, and mutants were screened for a reversion to snc1-like phenotypes. The isolated muse7 (mutant, snc1-enhancing, 7) mutant was shown to confer dwarf morphology, elevated expression of PATHOGENESIS-RELATED genes, and enhanced resistance to the virulent oomycete pathogen Hyaloperonospora arabidopsidis Noco2 when present in the mos4 snc1 background. Map-based cloning and Illumina whole genome sequencing revealed that the muse7 phenotypes are associated with a mutation in At5g46020, which encodes a protein of unknown function. This protein is conserved across most eukaryotes but is not present in Saccharomyces cerevisiae or Schizosaccharomyces pombe. Both the muse7-1 allele isolated from this screen and the muse7-2 exonic T-DNA insertion allele displayed enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, but not to P.s.t. DC3000 expressing the effector proteins AvrRpm1, AvrRpt2, or AvrRps4. While transcription of SNC1 is not elevated in the muse7 mutants, SNC1 protein accumulates in both alleles. Although proteasome-mediated 84  degradation is a well-studied event in immune regulation, no interactions were detected between MUSE7 and known components of this pathway, suggesting that MUSE7 may regulate SNC1 at the translational level. This study has demonstrated a novel role for MUSE7 in modulating plant immune responses, and may benefit future studies of MUSE7 homologs in other species.  4.2 Introduction  The plant immune system is subject to tight regulation, enabling these sessile organisms to ward off infection by most pathogenic microorganisms. As an initial line of defense, plants possess many physical and chemical barriers that hinder microbial access to plant cells; these barriers include the cuticle, the cell wall, and anti-microbial enzymes. However, the relationship between plants and phytopathogens is highly dynamic, and these defenses are sometimes breached. In case of such events, plants also possess a sensitive surveillance system that detects the presence of invading pathogens. Receptors on the cell surface are able to perceive conserved pathogen-associated molecular patterns (PAMPs) (Macho & Zipfel 2014). Via complex signaling pathways, this recognition leads to the induction of PAMP-triggered immunity (PTI) (Bigeard et al. 2015), which is mediated by a mitogen-activated protein kinase signaling cascade and results in a number of defense response outputs, including callose deposition to strengthen the cell wall, production of reactive oxygen species (ROS), and accumulation of the defense hormone salicylic acid (SA) (Hammond-Kosack & Jones 1996). In turn, however, many successful pathogens are able to deliver molecules (termed effectors) into the plant cell to inhibit PTI and promote infection. Escalating this “arms race”, higher plants have evolved a suite of intracellular immune receptor proteins that are able to detect effector molecules, either through direct protein-protein 85  interactions or indirectly through the perception of effector activities within the plant cell. These plant proteins are referred to as NOD-like receptors (NLRs) due to their resemblance to metazoan nucleotide-binding oligomerization domain (NOD)-containing proteins, which function as PAMP receptors in animals (Li et al. 2015). The majority of plant NLR proteins possess a central nucleotide-binding (NB) domain and a C-terminal leucine rich-repeat (LRR) domain. They can be subdivided into two classes based upon their different N-termini: some possess a Toll-interleukin1 receptor (TIR) domain and are thus termed TNLs, and others have a coiled-coil (CC) domain and are therefore referred to as CNLs. In the absence of pathogen detection, NLR proteins are expressed at low levels. Following effector recognition, they become activated and initiate a downstream signaling cascade that results in effector-triggered immunity (ETI). This type of immunity is typified by a stronger, faster, and more robust induction of the defense outputs that characterize PTI (Cui et al. 2015). ETI may also lead to a type of localized cell death referred to as the hypersensitive response (HR). Additionally, the immunity mediated by NLR proteins is subject to a positive transcriptional feedback defense amplification, whereby NLR protein activation results in transcriptional reprogramming that subsequently upregulates the expression of a number of defense-related genes, including many that encode NLR proteins themselves (Tsuda & Somssich 2015). These processes must be finely tuned, as there is a trade-off between plant growth and plant immunity; dwarfism and reduced viability are associated with precocious activation of immune responses. The snc1 autoimmune mutant has proven to be a useful tool in disentangling the regulatory events that govern NLR-mediated immunity. This mutant contains a gain-of-function mutation in the linker region between the NB and LRR domains of SNC1, a TNL protein (Li et 86  al. 2001; Zhang et al. 2003). Plants with this mutation are dwarf and have a distinct twisted leaf morphological phenotype. A previous screen for suppressors of snc1 yielded a number of novel positive regulators of plant immunity (reviewed in Johnson et al. 2012). Results from the MODIFIERS OF SNC1 (MOS) screen highlighted the importance of modulated transcription, RNA processing, nucleocytoplasmic trafficking, and protein modifications as key regulatory events in immunity. Based on the success of the MOS screen, a screen for enhancers of snc1 was performed in order to search for novel negative regulators of NLR-mediated immunity. The MUTANT, SNC1-ENHANCING (MUSE) screen was conducted using seeds from mos4 snc1, which is approximately wild-type-like in terms of both morphology and resistance (Palma et al. 2007). This mutant background was utilized in order to avoid potential lethality resulting from severe dwarfism caused by enhancer mutations. Mutants were screened for a reversion back to snc1-like phenotypes, and twelve mutant lines were selected for further characterization. A number of reports on muse mutants have been recently published. Approximately half of the characterized MUSE proteins were shown to play essential roles in NLR protein turnover (Huang et al. 2014a; Huang et al. 2014b; Xu et al. 2015; Huang et al. 2016). One MUSE gene was found to encode AtPAM16, a component of the protein import motor in the mitochondrial inner membrane that is required for the negative regulation of ROS production (Huang et al. 2013). Another MUSE gene encodes SPLAYED, a SWI/SNF chromatin remodeler that had previously been found to positively regulate immunity to necrotrophic pathogens (Johnson et al. 2015). Together, these reports demonstrate the efficacy of the MUSE screen in identifying molecular events in plant immunity. In this study, we report the characterization of muse7, which fully restores snc1-like resistance in the mos4 snc1 background.   87  4.3 Results  4.3.1 The muse7 mutation re-establishes snc1-like phenotypes in the mos4 snc1 background The muse7 mutant line was originally isolated from a forward genetic screen designed to identify snc1 enhancers in the mos4 snc1 genetic background, which was previously described (Huang et al. 2013). Morphologically, muse7 mos4 snc1 plants are similar to snc1 plants in that they are dwarf and have slightly twisted leaves (Figure 4.1A). The triple mutant also displays enhanced defense marker PATHOGENESIS-RELATED (PR) gene expression as compared to mos4 snc1 (Figures 4.1B-C), suggesting that the immune responses are partially activated in muse7 mos4 snc1 even in the absence of pathogens. Additionally, the triple mutant exhibits enhanced resistance to the virulent oomycete pathogen Hyaloperonospora arabidopsidis (H.a.) Noco2 as compared to mos4 snc1 (Figure 4.1D). Altogether these data show that the muse7 mutation reconstitutes snc1-like phenotypes in the mos4 snc1 background, indicating that muse7 is an enhancer of snc1.  88   Figure 4.1. Phenotypic characterization of the muse7 mos4 snc1. (A) Morphology of soil-grown wild-type, snc1, mos4 snc1, and muse7 mos4 snc1 plants. Photographs were taken four weeks post-germination. (B) PR2 gene expression, visualized using the GUS reporter gene assay. All genotypes contain a construct in which the promoter region of PR2 is fused to the coding sequence of β-glucuronidase (GUS), and following incubation with the substrate X-Gluc the presence and intensity of blue staining provides an indication of gene expression. Plants were grown for 10 days on MS media. (C) Endogenous expression of PR1 and PR2 relative to ACTIN7 as determined by reverse-transcription quantitative PCR using 30 cycles.  (D) Growth of H.a. Noco2 on indicated genotypes seven days post-inoculation with 1x105 spores/mL. Values represent the average of four replicates of five plants each ± SD.  4.3.2 MUSE7 encodes an uncharacterized protein conserved amongst eukaryotes To identify the molecular lesion responsible for the phenotypes associated with muse7, a positional cloning strategy was employed. The muse7 mos4 snc1 triple mutant, which was 89  generated in the Col-0 background, was crossed with wild-type Landsberg erecta (Ler). 24 F2 plants displaying the original triple mutant morphology were selected for linkage analysis. Insertion/deletion DNA markers generated using known polymorphisms between the Col-0 and Ler ecotypes were used to search for genomic regions with a strong linkage to the Col-0 genotype. Course mapping indicated that the muse7 mutation is located on chromosome 5 between 17.3 and 19.9 MB (Figure 4.2A). Fine mapping using approximately 500 segregating F3 plants narrowed down the location of the muse7 mutation to the region between 18.6 and 18.7 MB, at which point Illumina whole genome sequencing was performed to identify genes within this region containing mutations consistent with EMS mutagenesis. The only candidate gene identified was At5g46020, which contains a C to T point mutation resulting in a premature stop codon at Q121 (Figure 4.2B-C).   90   Figure 4.2. Positional cloning of MUSE7. (A) A genetic map of the region of chromosome 5 containing the MUSE7 locus, with markers used for mapping indicated. (B) Gene structure of MUSE7 and the position of the molecular lesions in muse7-1 (an EMS allele) and muse7-2 (an exonic T-DNA insertion allele). Boxes and lines represent exons and introns, respectively. Start and stop codons are indicated. (C) Sequence comparison between wild-type MUSE7 and muse7-1. Nucleotide substitution, indicated with a lower case ‘t’, results in a change from Q121 to a stop codon.  (D) Four-week-old soil-grown plants of the genotypes noted. MUSE7-GFP#1 and MUSE7-GFP#2 are from two independent transgenic complementing lines, where MUSE7 tagged with GFP and under the control of its native promoter was expressed in the muse7 mos4 snc1 background. Bar indicates 1 cm.  91  To verify that this mutation in At5g46020 is responsible for the muse7 phenotypes, transgene complementation was performed. A wild-type copy of the gene under the control of its native promoter was transformed into the muse7 snc1 mos4 triple mutant, and a reversion back to mos4 snc1-like morphology was observed (Figure 4.2D), indicating that the correct gene was cloned. As AT5G46020 has not been previously characterized, we have designated this locus as MUSE7 and the mutant allele identified in our screen as muse7-1.  MUSE7 is a single copy gene in Arabidopsis, and orthologs are present in all examined land plants in low copy number (Table 4.1). The protein encoded by MUSE7 possesses a conserved phosphoprotein PP28 domain (Figure 4.3). Using amino acid sequences for BLAST analysis, it was determined that MUSE7 homologs exist in all examined plants and animals as well as in many fungi, but not in either Saccharomyces cerevisiae or Schizosaccharomyces pombe. Overall, this suggests that MUSE7 is a highly conserved protein particularly among multicellular organisms, and that it likely functions in a conserved biological process.  Table 4.1. MUSE7 homologs are present in low copy number across all examined land plants. USEARCH was employed to identify all potential homologs.  92                             10         20         30         40         50              A.thaliana       MGRGKFKGKP TGQ-RRFSSA ADILAGTSAA RPRSFKQKEA EYEED-----  O.sativa         MGRGKFKGKP TGR-RNFSTP EEIAAGTSG- RPRTFKKNLA EEEKE-----  Z.mays           MGRGKFKGKP TGR-RNFSTP EEIAAGTSG- RPRTFKK--K EEEED-----  M.musculus       MPKGGRKGGH KGRVRQYTSP EEIDAQLQAE KQKANEEDEQ EEGGDGAS--  H.sapiens        MPKGGRKGGH KGRARQYTSP EEIDAQLQAE KQKAREEEEQ KEGGDGAA--  D.melanogaster   MPRGKFVN-H KGRSRHFTSP EELQQESEED SDQTSGSGSD SDDKDAAGGK                   * :*   .   .*: *.:::.  ::    .     ::  .    .   :                                 60         70         80         90        100             A.thaliana       ---------- ---------- ---------- --VEEESEEE SEEE-SEDEA  O.sativa         ---------- ---------- ---------- --EEEDDIEE SEEEESEDES  Z.mays           ---------- ---------- ---------- --EEEVEREE SEEE-SEEDS  M.musculus       ---------- ---------- ---------G DPKKEKKSLD SDES-EDEDD  H.sapiens        ---------- ---------- ---------G DPKKEKKSLD SDES-EDEED  D.melanogaster   ASSSASKAKA PATRKAPVNR NQKSRSAAGA GAASSSESES GEDSDDDSEA                                                       .. .  . .::. .:.:                            110        120        130        140        150         A.thaliana       D--VKKKGAE AVIEVDNPNR VRQKT---LK AKDLDASKT- ------TELS  O.sativa         EGKAKHKGTE GLIQIENPNL VKAKN---IK AKEVDLGKT- ------TELS  Z.mays           DEKTKHKGTE GIIQIENPNL VKAKN---IK AKEVDFGKT- ------TELS  M.musculus       DYQQKRKGVE GLIDIENPNR VAQTT---KK VTQLDLDGP- ------KELS  H.sapiens        DYQQKRKGVE GLIDIENPNR VAQTT---KK VTQLDLDGP- ------KELS  D.melanogaster   EARDAKKGVA SLIEIENPNR VTKKATQKLS AIKLDDGPAG AGGNPKPELS                   :    :**.  .:*:::***  *  .     . . .:* . .         ***                           160        170        180        190        200         A.thaliana       RREREELEKQ RAHERYMRLQ EQGKTEQARK DLDRLALIRQ QREEAAKKRE  O.sativa         RREREEIEKQ KAHERYMKLQ EQGKTEQARK DLERLALIRQ QRADAAKKRE  Z.mays           RREREELEKQ KAHERYMKLQ EQGKTEQARK DLERLALIRQ QRADAAKKRE  M.musculus       RREREEIEKQ KAKERYMKMH LAGKTEQAKA DLARLAIIRK QREEAARKKE  H.sapiens        RREREEIEKQ KAKERYMKMH LAGKTEQAKA DLARLAIIRK QREEAARKKE  D.melanogaster   RREREQIEKQ RARQRYEKLH AAGKTTEAKA DLARLALIRQ QREEAAAKRE                   *****::*** :*::** :::   *** :*:  ** ***:**: ** :** *:*                           210        220     A.thaliana       EEKAARDA-K KVEGRK---- ---- O.sativa         EEKAAKEQ-R KAEARK---- ---- Z.mays           EEKAAKEQ-R KSEARK---- ---- M.musculus       EERKAKDD-A TLSGKRMQSL SLNK H.sapiens        EERKAKDD-A TLSGKRMQSL SLNK D.melanogaster   AEKKAADVGT KKPGAK---- ----                   *: * :    .  . :           Figure 4.3. Multiple alignment of MUSE7 homolog amino acid sequences. MUSE7 is highly conserved in higher plants and animals. Dashes indicate alignment gaps; “*” indicates identical residues; “:” indicates conserved substitutions; “.” indicates semi-conserved substitutions. Red font indicates location of casein kinase substrate, phosphoprotein PP28 domain. Highlighted locations: green – known phosphorylated serines in Rattus norvegicus, shown in the M. musculus homolog (Shen et al. 1996); yellow – muse7-1 mutation.  93  4.3.3 Two independent muse7 single mutant lines exhibit enhanced disease resistance To assess how immune responses are altered in the muse7 single mutant, muse7-1 and muse7-2 (an exonic T-DNA insertion allele) were characterized. Apart from the same slightly dwarf, rounded-leaf morphology (Figure 4.4A), both mutant lines were developmentally wild-type-like (Figure 4.5). Upon infection by the virulent bacterial strain Pseudomonas syringae pv. tomato (P.s.t.) DC3000, both muse7 alleles exhibit enhanced resistance compared with wild-type (Figures 5.4B), although the resistance displayed by muse7-2 is significantly stronger than that observed for muse7-1. When treated with the virulent oomycete H.a. Noco2 both alleles showed a general trend of enhanced resistance but only muse7-2 was consistently different from wild-type (Figure 4.4C). When these two mutant alleles were challenged with the avirulent bacteria P.s.t. DC3000 expressing the effector proteins AvrRpt2, AvrRpt4, or AvrRpm1, respectively, no significant difference in resistance compared with wild-type was observed (Figures 4.4D-F). These data suggest that MUSE7 serves as a negative regulator of immunity.  4.3.4 MUSE7 localizes to both the nucleus and the cytoplasm To gather clues as to the potential function of MUSE7, its subcellular localization was examined by transforming muse7 mos4 snc1 plants with a construct containing MUSE7 expressed using the native promoter and possessing a C-terminal GFP tag. The leaves and roots of two independent lines homozygous for single-copy transgene insertion were examined using confocal microscopy. GFP fluorescence was observed in both the nucleus and the cytoplasm in both tissue types (Figure 4.6), indicating that MUSE7 has a broad subcellular distribution. However, the MUSE7 protein has a predicted size of 18.9 kDa, thus we cannot exclude the possibility that it may freely diffuse into the nucleus. 94   Figure 4.4. Characterization of two independent muse7 single mutant alleles. (A) Morphological phenotypes of four-week-old soil-grown wild-type, muse7-1, and muse7-2 plants. Bar represents 1 cm. (B) Growth of P.s.t. DC3000 on wild-type, muse7-1, and muse7-2 plants three days post-infiltration. Values represent the average of five replicates ± SD. Letters indicate statistical difference (single-factor ANOVA and Tukey-Kramer post-hoc analysis). (C) Growth of H.a. Noco2 on wild-type, snc1, muse7-1, and muse7-2 plants seven days post-infection. Values presented are averages of three replicates ± standard deviation. Letters indicate statistical difference (single-factor ANOVA and Tukey-Kramer post-hoc analysis). (D-F) Growth of (D) P.s.t. DC3000 AvrRpt2 (E) P.s.t. DC3000 AvrRpt4, and (F) P.s.t. DC3000 AvrRptm1 on indicated genotypes three days post-infiltration. Values represent the average of five replicates ± SD.  95   Figure 4.5. Characterization of muse7 developmental phenotypes. (A) Primary root lengths of wild-type, muse7-1, and muse7-2 seedlings were measured to the nearest millimeter 10 DAG. Values presented are averages of four seedlings per genotype ± standard deviation, and are representative of values obtained in three biological replicates. Four wild-type seeds and four mutant seeds of one of the two muse7 genotypes (eight seeds total) were sown on ½ MS media plates, and seedlings were grown vertically. (B) Wild-type and muse7-1 seedlings described in (A). (C, D) Rosette leaves were counted for each genotype when the shoot was 6 - 10 cm. Plants were grown under (C) long day (16h light/8h dark) or (D) short day (8h light/16h dark) conditions. Values presented are the averages of 12 plants per genotype ± standard deviation. (E) Approximately six-week-old wild-type and muse7 plants grown under long day conditions. No obvious differences in height or silique distribution are apparent. (F) Five siliques selected from mid-shoot were collected from three plants per genotype and measured to the nearest millimeter. Values presented are averages ± standard deviation. 96   Figure 4.6. Subcellular localization of MUSE7-GFP. MUSE7-GFP fluorescence in Arabidopsis leaf and root cells as observed by confocal microscopy. Experiments were repeated with multiple cells from independent MUSE7::MUSE7-GFP complementing lines (Figure 5.2D). Scale bars represent 10 µm. C: cytoplasm; CS: cytoplasmic strand; N: nucleus.  4.3.5 Mutations in MUSE7 affect SNC1 accumulation  As the genesis of this project was a screen for enhancers of snc1, muse7 snc1 double mutants were generated to examine the enhancing effect of muse7 in the absence of mos4. Strikingly, both muse7 snc1 lines exhibit severe dwarfism (Figure 4.7A). This enhancement of snc1-like phenotypes in the double mutants is likely the result of misregulation of SNC1 at either (i) the 97  transcriptional level, or (ii) the protein level. Using qPCR, it was determined that SNC1 expression is similar in muse7-2 snc1 as compared to snc1 (Figure 4.7B), suggesting that MUSE7 does not regulate SNC1 at the transcriptional level. SNC1 protein levels were assessed using western blotting and while there was no observable difference between wild-type and muse7-2, a significant accumulation of the protein was observed in muse7-2 snc1 as compared to snc1 (Figure 4.7C-E). These data reveal that MUSE7 is involved in the negative regulation of SNC1 protein accumulation, as the loss of MUSE7 results in a higher level of SNC1 protein.    Figure 4.7. Regulation of SNC1 by MUSE7. (A) Four-week-old wild-type, snc1, muse7-1, muse7-2, muse7-1 snc1, and muse7-2 snc1 soil-grown plants. Bar indicates 1 cm. 98  (B) Endogenous expression of SNC1 relative to ACTIN7 in 21-day-old wild-type, snc1, muse7-2, and muse7-2 snc1 soil-grown seedlings. Values presented are averages of three replicates ± standard deviation. Letters indicate statistical difference (Each pair, Student’s t test, p-value < 0.05). (C) Western blot analysis of SNC1 protein levels in total protein extracts from 21-day-old wild-type, snc1, muse7-2, and muse7-2 snc1 soil-grown seedlings. (D) Image J analysis of SNC1 protein levels in wild-type and muse7-2. Values presented are the average of five blots ± SD. (E) Image J analysis of SNC1 protein levels in snc1 and muse7-2 snc1. Values presented are the average of three blots ± SD. Significant differences are indicated by asterisks (** p-value < 0.01).  4.3.6 MUSE7 does not appear to interact with known regulators of SNC1 turnover While the previous experiments indicate that MUSE7 regulates SNC1 at the protein level, it is unclear whether this regulation occurs at the stage of protein biosynthesis or through protein degradation. Recent reports on other MUSEs have identified a number of proteins that are involved in 26S-proteosome-mediated turnover of NLR proteins, highlighting the importance of this biological process in facilitating immune regulation. Co-immunoprecipitation experiments were therefore performed in Nicotiana benthamiana in order to determine if MUSE7 directly interacts with proteins known to play a role in NLR degradation (including HSP90.3 and CPR1) or with SNC1. However, no interactions were detected (Figure 4.8). These results indicate that MUSE7 likely regulates SNC1 protein levels either at the point of protein biosynthesis or via a previously uncharacterized degradation event.    99   Figure 4.8. MUSE7 does not coimmunoprecipitate with HSP90.3, CPR1, or SNC1 following transient co-expression in Nicotiana benthamiana. Co-immunoprecipitation assays were conducted using MUSE7-FLAG and (A) HSP90.3-HA, (B) CPR1-HA, and (C) SNC1-HA. All immunoprecipitations were performed 48 h following transient co-expression in N. benthamiana using anti-FLAG beads, and immunoblotting was performed using antibodies against HA and FLAG, respectively.   4.4 Discussion In this study, the previously uncharacterized Arabidopsis protein MUSE7 was shown to be a negative regulator of NLR protein accumulation. Little can be inferred about the function of MUSE7 based on its homologs in other eukaryotes. The closest human homolog to MUSE7 is PDAP1 (PLATELET-DERIVED GROWTH FACTOR (PDGF)-ASSOCIATED PROTEIN 1), which binds to two different isoforms of PDGF and modulates their mitogenic activities (Fischer & Schubert 1996). However, the Arabidopsis genome does not encode any PDGF homologs, thus this function does not seem to be conserved across kingdoms. The Rattus norvegicus homolog of MUSE7 was previously shown to be phosphorylated by casein kinase II at S62 and S59, sequentially (Shen et al. 1996; Figure 4.3). This suggests that MUSE7 function may be modulated by phosphorylation. Although there is currently no empirical evidence to support this postulation, future experiments using phosphomimetic mutant versions of the MUSE7 protein may be useful in testing this hypothesis. The results presented in this study provide the first indication as to the biological role of MUSE7 in plants. 100  MUSE7 was isolated from a screen for enhancers of the autoimmune mutant snc1, suggesting that it is involved in the negative regulation of plant innate immunity (Figure 4.1). Consistently, both muse7 mutant alleles showed enhanced resistance to P.s.t. DC3000 (Figure 4.4). However, both mutant lines show wild-type-like resistance to P.s.t. DC3000 that has been modified to express only AvrRpt2, AvrRpt4, or AvrRpm1 effectors, respectively. This result indicates that the resistance in muse7 single mutants is not predominantly mediated by RPS2, RPS4, or RPM1. The NLR protein SNC1 was found to accumulate in the muse7 snc1 double mutant as compared to snc1 (Figure 4.7). An examination of the accumulation of other NLR proteins in muse7 plants would provide insight into whether the effects of MUSE7 on protein levels are specific to SNC1.  The increased level of SNC1 protein in muse7 snc1 relative to snc1 did not correlate with elevated SNC1 transcription (Figure 4.7). This implies that MUSE7 likely functions to negatively regulate either the synthesis or degradation of NLR proteins. The degradation of NLR proteins via the 26S proteasome is a key regulatory step in plant immunity, and recent reports have implicated a number of proteins in this process. To determine whether MUSE7 is also part of the 26S proteasome-mediated degradation pathway, co-immunoprecipitation assays were performed to ascertain whether it interacts with known components of this process. However, no interaction was detected between MUSE7 and CPR1, HSP90.3, or SNC1 (Figure 4.8). This is not an exhaustive list of the components that function in proteasome-mediated NLR protein degradation, and it is possible that MUSE7 interacts specifically and exclusively with untested protein(s) (e.g. SGT1, SRFR1, CUL1, etc.). Further experimentation is required to definitively rule out this possibility. It is also conceivable that MUSE7 may contribute to the degradation of 101  NLR proteins through a novel, uncharacterized proteasome-independent pathway, although this is unlikely and would be difficult to verify. A more likely prospect is that MUSE7 functions to suppress NLR protein biosynthesis. In a transcriptional analysis of leaf-expressed genes in Arabidopsis, the expression profile of MUSE7 was found to correlate closely with a number of genes involved in protein biosynthesis (Street et al. 2008). Also of note, human PDAP1 was identified as a candidate RNA-binding protein as part of a large-scale quantitative proteomics analysis (Baltz et al. 2012). It is conceivable that MUSE7 binds to mRNA transcripts and affects translation by the protein biosynthesis machinery. Future experiments assaying whether MUSE7 interacts with ribosomal proteins will be useful in testing this hypothesis. If MUSE7 is found to function as a regulator of protein synthesis, two models for its function are conceivable: (i) MUSE7 binds to transcripts encoding NLRs and represses their ability to be translated, or (ii) MUSE7 binds to non-NLR-encoding transcripts and actively recruits the protein synthesis machinery, such that these other transcripts are preferentially translated over NLR-encoding mRNAs. RNA immunoprecipitation and sequencing (RIP-seq) could be performed to determine if MUSE7 has RNA-binding activity, and if so, which sequences are bound. Overall, our results show that we have identified a novel protein involved in plant immunity. This protein negatively regulates NLR protein levels, either in terms of protein biosynthesis or as part of an uncharacterized degradation pathway. The work presented here furthers our understanding of the regulation of NLR homeostasis and may serve to inform studies in other species, as the MUSE7 homologs across eukaryotes have not been well-characterized.  102  4.5 Methods and materials  4.5.1 Plant growth conditions and mutant isolation Plants were grown either on soil or on half-strength Murashige and Skoog (MS) media supplemented with 1% sucrose and 0.3% phytagel. Plants were grown under long day conditions (16 h light/8 h dark) at 22ºC in climate-controlled chambers unless short day conditions are indicated, in which case 12 h light/12 h dark settings were used.   4.5.2 Positional cloning The muse7 mos4 snc1 triple mutant, which was generated in the Col-0 background, was crossed with wild-type Landsberg erecta (Ler). Approximately 300 F2 seeds were planted and DNA was extracted from 24 mutant plants displaying the original triple mutant morphology. Insertion/deletion DNA markers were generated using the Monsanto Arabidopsis polymorphisms and Landsberg sequence collections (Jander et al. 2002) obtained from The Arabidopsis Information Resource (TAIR; www.arabidopsis.org). These markers were used to search for genomic regions showing a strong linkage with the Col-0 genotype. Once linkage was established, approximately 500 F3 plants segregating for the muse7 mutation but homozygous for mos4 and snc1 were used for fine mapping. After narrowing down the location of the mutation to between 18.6 and 18.7 MB on chromosome 5, genomic DNA extracted from plants homozygous Col-0 for the region containing the muse7 mutation was analyzed using the Illumina sequencing platform.   103  4.5.3 Total RNA extraction and analysis Approximately 0.1 g tissue was collected from 2-week-old seedlings grown on ½ MS. Total RNA was extracted using the Totally RNA Kit (Invitrogen), and 0.4 μg RNA was reverse transcribed to cDNA using the Reverse Transcriptase M-MLV (Takara). Primers used for amplification of SNC1 and ACTIN7 were previously described (Zhang et al., 2003; Xu et al., 2012).  4.5.4 Infection assays Spray inoculation of H.a. Noco2 was performed using a solution with a concentration of 105 spores per mL of water. Seedlings were grown for 10 d prior to inoculation, and afterwards were grown at 18ºC in a growth chamber with ~80% humidity and a 12 h light/12 h dark cycle. After 7 d, five plants were shaken in 1 mL of water and a hemocytometer was used to quantify spore growth. Three trials were performed, and for each trial five replicates were included. For bacterial infections, a needle-less syringe was used to infiltrate the leaves of 4-week-old soil-grown plants. Bacterial suspensions (OD600=0.001) in 10 mM MgCl2 were used. Leaf discs of uniform size were harvested from infected leaves at day 0 and day 3 and were ground in 10 mM MgCl2 (200µL on day 0; 500µL on day 3), and serial dilutions were performed and plated on LB media with streptomycin selection. Colony forming units were quantified after incubating plates at 28ºC for 24 h.   104  4.5.5 Preparation of transgene constructs and plant transformation  The full length At5g46020 genomic sequence, including 952 bp upstream of the start codon, was amplified via PCR, cloned into the pCambia1305 vector (versions both with and without a C-terminal GFP tag were used), and transformed into muse7 mos4 snc1 plants using the floral dip method (Clough and Bent, 1998). Transgenic plants were selected for on ½ MS plates containing 50 mg/mL hygromycin. For transient protein expression in Nicotiana benthamiana, the coding sequence of MUSE7 was cloned into a modified pCambia1305 vector containing a 35S promoter region and a C-terminal FLAG tag. For transformation of 3-week-old N. benthamiana, construct-containing Agrobacterium was cultured overnight at 28°C in liquid LB media containing 50 µg/mL kanamycin. The overnight culture was then diluted (1:50) in resuspension media [10.5 g/L K2HPO4, 4.5 g/L KH2PO4, 0.5 g/L sodium citrate, 1.0 g/L (NH4)2SO4, 0.2% glucose, 0.5% glycerol, 1 mM MgSO4, 50 µM acetosyringone and 10 mM N-morpholino-ethanesulfonic acid (MES) pH 5.6], and incubated at 28°C for another 5-6 h. The bacteria were then pelleted by centrifugation at 4,000 RPM  for 10 min and resuspended in MS buffer (4.4 g/L MS, 10 mM MES, 150 µM acetosyringone). For infiltration, each bacterial strain was diluted as follows: 35S::HSP90.3-HA (OD600=0.4), MUSE13::MUSE13-GFP (OD600=0.4), 35S::SNC1-HA (OD600=0.2), 35S::MUSE7-FLAG (OD600=0.3), and 35S::CPR1-FLAG (OD600=0.3).   4.5.6 Protein extraction and co-immunoprecipitation For extracting total protein from Arabidopsis plants, the protocol outlined in Tsugama et al. 2011 was employed. Briefly, 50 mg of aerial tissue was harvested from 3-week-old soil-grown plants. Tissue was then boiled in SDS buffer [0.1M EDTA, pH 8.0; 0.12M Tris-HCL, pH 6.8; 4% w/v 105  SDS; 10% v/v β-mercaptoethanol; 5% v/v glycerol; 0.005% w/v bromophenol blue] for 10 min before separating samples on SDS-PAGE gels. A SNC1-specific peptide (KAKSEDEKQS) was used to generate an anti-SNC1 antibody. Co-immunoprecipitation of proteins transiently expressed in N. benthamiana was performed at 4°C. Approximately 1.5 g of leaf tissue was used for both the control (leaves only transformed with the prey) and the sample (leaves transformed with both bait and prey), respectively. Tissue was ground into fine power using liquid nitrogen and a pre-chilled mortar and pestle, and 2.5 w/v extraction buffer [50 mM Tris-HCl pH 7.5; 150 mM NaCl; 10 mM EDTA; 1 mM EGTA; 0.15% Nonidet P-40 substitute; 10% glycerol; 1 mM DTT; 2 mM NaF; 1 mM Na2MoO4·2H2O; 2% w/v polyvinylpolypyrrolidone; 1 mM PMSF; 1× protease inhibitor cocktail] was added to each sample. Samples were centrifuged (14,000 RPM, 10 min) and the supernatant was collected. Proteins were immunoprecipitated using 30 µL anti-FLAG M2 beads (Sigma; Cat. #A2220); samples were incubated for 3 h at 4°C. Beads were then washed three times using wash buffer [50 mM Tris-HCl pH 7.5; 150 mM NaCl; 10 mM EDTA; 1 mM EGTA; 0.15% Nonidet P-40 substitute; 10% glycerol; 1 mM DTT; 2 mM NaF; 1 mM Na2MoO4·2H2O; 1 mM PMSF; 1× protease inhibitor cocktail]. Beads were next incubated with 3XFLAG peptide for 1 h at 4°C, then centrifuged (4,000 RPM, 2 min). The supernatant was combined with 2X SDS loading buffer, and samples were incubated at 95°C for 10 min before separation on 10% SDS-PAGE gels. 106  Chapter 5: Final perspectives  5.1 Overview  An important layer of the plant immune system is constituted by a suite of intracellular NOD-like receptor (NLR) proteins that recognize pathogenic effector molecules and subsequently initiate signaling cascades that lead to the induction of defense responses (Li et al. 2015). While the outputs of immune signaling are fairly well-characterized, the means by which these signaling pathways are adjusted and controlled are less defined. By using the autoimmune gain-of-function mutant snc1 (suppressor of npr1, constitutive 1) (Li et al. 2001; Zhang et al. 2003), the previously reported MODIFIER OF SNC1 (MOS) screen was able to successfully isolate a number of novel positive regulators of plant immunity. The MOS proteins were shown to regulate SNC1-mediated immunity via epigenetic regulation (MOS1 [Li et al. 2010; Li et al. 2011]; MOS9 [Xia et al. 2013]), transcriptional repression (MOS10 [Zhu et al. 2010]), RNA processing (MOS2 [Zhang et al. 2005]; MOS4 [Palma et al. 2007]; MOS12 [Xu et al. 2012]), mRNA export (MOS3 [Zhang & Li 2005]; MOS14 [Xu et al. 2011]), nucleocytoplasmic protein trafficking (MOS6 [Palma et al. 2005]; MOS7 [Cheng et al. 2009]; MOS11 [Germain et al. 2010]), and post-translational protein modifications (MOS5 [Goritschnig et al. 2007]; MOS8 [Goritschnig et al. 2008]). Based on the success of the MOS screen, a MUTANT, snc1-ENHANCING (MUSE) screen was conducted to identify negative regulators of innate immunity. Recently, the characterizations of several MUSE genes have been published. The encoded proteins have demonstrated roles in NLR protein turnover (MUSE3 [Huang et al. 2014]; MUSE6 [Xu et al. 107  2015]; MUSE10 and MUSE12 [HSP90s; Huang et al. 2014]; MUSE13 and MUSE14 [Huang et al. 2016]), as well as regulating the production of reactive oxygen species (MUSE5 [Huang et al. 2013]). Together, these reports clearly demonstrate that the strict control of NLR protein levels is crucial in the modulation of immune responses; however, the negative regulatory mechanisms underlying the plant immune system are expected to extend to other biological processes as well. The overarching objective of the research that constitutes this thesis was to identify novel regulators of NLR-mediated immune signaling, with the ultimate goal of furthering the collective understanding of the molecular mechanisms that control the timing and amplitude of immune response activation. For this thesis research, three uncharacterized muse mutant lines were selected for further study. The molecular lesions responsible for the observed mutant phenotypes were cloned, and the roles of these three genes in regulating immunity were examined. Based on the experimental findings described in this dissertation, the three MUSE proteins (MUSE4, MUSE7, and MUSE9) can be incorporated into the SNC1 regulatory model (Figure 5.1). The studies reported here have provided new insights into three different regulatory steps that are essential for immune response modulation. 108   Figure 5.1. A modified model depicting the involvement of the MOS proteins and three MUSE proteins in NLR protein-mediated defense signaling pathways in Arabidopsis, using SNC1 as an example of the journey of TNL proteins. 1. At chromosomal level, MOS1, ATXR7 and MOS9 up-regulate the transcription of SNC1 through chromatin remodeling, while MUSE9 (SPLAYED) negatively affects SNC1 transcription. 2. MOS2, MOS4, MOS12, and MUSE4 (NRPC7; an RNA Polymerase III subunit) are required for the proper splicing of the transcripts of SNC1. 3. The Nup107-160 complex and MOS11 play key roles in the export of total mRNA (including mature mRNA of SNC1), which is required for effective defense. 4. MOS5 is an E1 ubiquitin-activating enzyme, an essential component of the ubiquitination cascade, required for the regulation of defense signaling components. As an example, the SCFCPR1 E3 ubiquitin ligase complex targets SNC1 for degradation which prevents autoimmunity caused by over-accumulation of NLR proteins. MOS8 positively regulates plant defense, possibly through prenylation that affects the targeting of defense regulators. MUSE7 negatively regulates SNC1 protein accumulation. 5. MOS6 and MOS7 are involved in the nucleocytoplasmic shuttling of defense signaling molecules such as SNC1, EDS1, and NPR1. Like with RPS4, EDS1 is probably required for the nuclear localization and activation of SNC1 upon the recognition of its corresponding effector 109  (Bhattacharjee et al. 2011; Heidrich et al. 2011). MOS14 is required for the nuclear import of splicing factors that may affect defense regulator RNA processing. 6. MOS10 activates the SNC1-mediated defense through transcriptional repression of negative regulators of defense such as DND1 and DND2.  5.2 Immunoregulatory mechanisms examined in this thesis  5.2.1 Chromatin architecture and transcriptional modulation  5.2.1.1 Findings from the MUSE9/SPLAYED study The muse9 mutation enhances all examined snc1-associated phenotypes in the snc1 mos4 genetic background (Figure 2.1). The mutant was found to possess a point mutation in At2G28290 (Figure 2.2), which encodes the SWI/SNF chromatin remodeler SPLAYED (SYD). While the muse9/syd-10 single mutant did not display enhanced disease resistance against virulent pathogens, plants homozygous for the syd-4 mutant allele were significantly more resistant to the bacterial pathogen Pseudomonas syringae pv. maculicola (P.s.m.) ES4326 (Figure 2.4B). Both alleles are associated with the enhanced expression of PATHOGENESIS-RELATED (PR) genes and SNC1 but not RPP4, which is an NLR-encoding gene located proximal to SNC1 (Figure 2.4A, C). As chromatin remodelers are known to affect DNA methylation profiles, which in turn can affect gene expression, the methylation status surrounding the SNC1 locus was examined in the syd-4 mutant (Figure 2.7). While an overall decrease in CHH methylation was observed in the mutant background, analyses of global methylation mutants indicated that a decrease in methylation around the SNC1 locus does not always correspond with a decrease in SNC1 110  expression. This indicates that the increased SNC1 expression observed in the syd mutants is unlikely to be a result of the observed changes in methylation status at this locus. SYD was previously characterized as a positive regulator of jasmonate- and ethylene-mediated defense responses against necrotrophic pathogens (Walley et al. 2008). The study of muse9/syd-10 demonstrated a novel role for SYD in negatively regulating salicylic acid-mediated defense responses via modulation of SNC1 transcription. Therefore, it seems that SYD functions antagonistically to MOS1 and MOS9 in regulating transcription at this locus. This underscores the importance of fine-tuned transcriptional control of NLR protein-encoding loci in mediating immune responses.  5.2.1.2 Future directions Studies of SYD are limited by the large size of the gene (16,870 bp), which makes its cloning into a binary vector a difficult task and thus precludes most biochemical analyses. To date, none of the laboratories that work with this gene have overcome this obstacle. Further studies are required to more thoroughly examine the immunoregulatory role played by SYD. Specifically of interest is whether SYD directly represses transcription of SNC1, perhaps through interactions with other transcriptional activators or repressors that act at this locus, or whether the effect is more indirect (for example, SYD may regulate the transcription of a gene encoding a protein that in turn affects SNC1 transcription). Chromatin immunoprecipitation (ChIP) assays may be performed to determine whether SYD directly binds the SNC1 locus; this would require the use of a SYD-specific antibody which has been developed by the Wagner research group (Walley et al. 2008). Alternatively, if SYD was not found to associate with the SNC1 locus, a large-scale ChIP-sequencing approach could be employed. Using this technique, all DNA fragments bound 111  by SYD would be sequenced. This may yield a large number of results, as SYD is known to bind multiple loci (Kwon et al. 2005; Walley et al. 2008). After developing a catalogue of putative SYD-bound loci, single and double mutant analyses using candidate genes together with the snc1 mutant could be employed to determine if the candidate genes affect SNC1 transcription.  5.2.2 Alternative splicing of genes encoding NLR proteins  5.2.2.1 Findings from the MUSE4/NRPC7 study The muse4 mutant allele confers an enhancement of snc1-like phenotypes in the mos4 snc1 background (Figure 3.1). The mutation responsible for these observed phenotypes is located in an intron/exon splice site junction in AT1G06790, which encodes the RNA Polymerase (Pol) III subunit NRPC7 (Figure 3.2). The muse4/nrpc7-1 mutation results in the retention of an intron in some (but not all) transcripts produced from this locus (Figure 3.4), and is likely a partial loss-of-function allele as all previously reported Pol I, II, and III mutants are lethal. The nrpc7-1 mutation was likely isolated in our screen for snc1 enhancers due to the pleiotropic phenotypes of the mutant. The single mutant does not display increased SNC1 expression or protein accumulation (Figure 3.5), nor does it display enhanced resistance to virulent pathogens (Figure 3.6). However, the nrpc7-1 mos4 snc1 triple mutant shows an alteration in the alternative splicing patterns of both SNC1 and RPP4 (Figure 3.6). This may be due to the cumulative splicing defects associated with mutations in nrpc7-1 and mos4: NRPC7 is required for the transcription of the spliceosome component U6 (Figure 3.6), and MOS4 is an important component of a spliceosome-associated complex (Johnson et al. 2011).  112  Alternative splicing of the genes encoding NLR proteins has previously been shown to affect their function and is posited to be a means of honing immune responses (Jordan et al. 2002). The characterization of nrpc7-1 supports this hypothesis.  5.2.2.2 Future directions The defects associated with nrpc7-1 are broad, and the immune phenotypes observed in nrpc7-1 mos4 snc1 are likely a result of broad changes in transcription and splicing. As such, the results of this study do not suggest that Pol III plays a direct, targeted role in immune regulation, thus the applications of the nrpc7-1 mutant to examining plant immunity are limited. By virtue of being the first reported viable Pol III mutant, nrpc7-1 is likely to be of interest to researchers in the fields of transcription and RNA biology. For example, future studies could focus on using this mutant to examine the biogenesis of weakly characterized small RNAs. Although the suites of RNA molecules transcribed by the various Pols are largely known, there are some RNA molecules for which the biogenesis pathways are unclear. By looking at the accumulation of these RNAs in the nrpc7-1 background, some insight may be gained as to whether their transcription requires fully functional Pol III.   5.2.3 NLR protein accumulation  5.2.3.1 Findings from the MUSE7 study Similar to what was observed for muse9 and muse4, the muse7 mutation enhances snc1-like morphology and resistance in the mos4 snc1 genetic background (Figure 4.1). The corresponding molecular lesion is located in AT5G46020, which encodes a protein of unknown function (Figure 113  4.2). The annotation of this gene in TAIR (arabidopsis.org) states that it contains a casein kinase substrate phosphoprotein PP28 domain; this is based on its homology to a protein studied in Rattus norvegicus that is phosphorylated by casein kinase II (Shen et al. 1996). MUSE7 is conserved in higher plants and does not have any close homologs in the Arabidopsis thaliana genome (Figure 4.3).  While mutations in muse7 do not result in an elevation of SNC1 transcription, they do cause an increase in SNC1 protein levels (Figure 4.7). This indicates that MUSE7 either (i) contributes to the targeted degradation of NLR proteins, or (ii) negatively regulates their biosynthesis. In recent years, proteasome-mediated degradation of NLR proteins has emerged as a critical component of immune response regulation (Cheng et al. 2011; Huang et al. 2014a; Huang et al. 2014b; Huang et al. 2016). No interaction was detected between MUSE7 and CPR1 or HSP90.3, both of which are known regulators of NLR protein turnover (Figure 4.8). There was also no detectable interaction between MUSE7 and SNC1. These data, when taken together with a recent report that revealed that MUSE7 co-expresses with a number of protein biosynthesis genes (Street et al. 2008), indicate that MUSE7 may regulate SNC1 protein synthesis rather than degradation.  The study of MUSE7 has resulted in the identification of a novel regulator of NLR protein accumulation, and is the most thorough characterization of this gene in any species. However, the lack of functional domains in the MUSE7 protein has made analyses of this protein somewhat difficult.  114  5.2.3.2 Future directions Many potential avenues of future MUSE7 research exist. It will be important to determine whether MUSE7 affects the accumulation of NLR proteins other than SNC1, which can be done by examining the expression of tagged NLR proteins in the muse7 background as compared to wild type. It will also be of interest to determine whether MUSE7 is phosphorylated and if so whether this phosphorylation affects its function in immune regulation. To examine these possibilities, phosphomimetic mutant versions of the MUSE7 protein can be generated and their resistance phenotypes can be assayed. To investigate whether the phosphorylation status of MUSE7 is altered during infection, a band mobility shift assay could be employed. Importantly, future studies of MUSE7 should focus on identifying interactor protein(s) in order to clarify the functional role this protein plays in regulating NLR protein accumulation. Co-immunoprecipitation assays in N. benthamiana may be used to determine whether MUSE7 interacts with ribosomal proteins, as might be expected if MUSE7 does impact NLR protein synthesis. If no interactions are detected using a candidate-based approach, an immunoprecipitation/mass spectrometry (IP/MS) method may be employed. However, a potential disadvantage of this technique is the relatively high rate of false positives. Also, MUSE7 is expressed at a low level, and previous IP/MS attempts in our laboratory using weakly expressed proteins have been unsuccessful. A yeast two-hybrid screen may be an effective tool in identifying interacting proteins; however, MUSE7 has already been screened against a library of 8000 cDNAs, and no interactions were detected. Additionally, no candidate interactors are currently listed in the Arabidopsis thaliana CCSB Interactome Database (interactome.dfci.harvard.edu/A_thaliana/). 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