"Science, Faculty of"@en . "Botany, Department of"@en . "DSpace"@en . "UBCV"@en . "Wu, Di"@en . "2023-01-31T08:00:00Z"@en . "2020"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "Plants employ a multi-layered innate immune system to ward off invading pathogens. The pattern-recognition receptors (PRRs) play pivotal roles in immune signaling by recognizing the pathogen-derived conserved molecular signatures termed PAMPs (pathogen-associated molecular patterns). Finetuning the activity of PRR signaling is vital for the well-being of plants, as the perturbation of components of the PRR complex typically causes disastrous outcomes on growth and defense. However, how plants achieve such tight regulation of the PRR signaling is not fully understood. BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), which functions as a coreceptor for PAMP recognition, is an essential component of the PRR complex. BAK1 harbors a mysterious carboxyl-terminal tail (CT) beyond its kinase domain. In the present study, we clarified the biological significance of this CT region using a CT-deletion bak1 mutant allele. Our data showed that the CT is required for BAK1\u00E2\u0080\u0099s function in PAMP-triggered immunity (PTI), but is dispensable for cell death control and brassinosteroid signaling, indicating the differential requirement of the CT in development and immunity. In vitro phosphorylation assay revealed that the CT is required for the autophosphorylation activity of BAK1, suggesting that the CT serves as an intrinsic modulator to promote BAK1\u00E2\u0080\u0099s kinase activity. Plant metacaspases (MCs) are structural homologs of mammalian caspases. Arabidopsis METACASPASE 2 (MC2) carries an N-terminal prodomain, whose function is unclear. Here, we uncovered an imperative role of the MC2 prodomain in activating PTI signaling via genetic analysis of an mc2 mutant allele called mc2-1. Heightened expression of the MC2 prodomain in mc2-1 results in constitutive activation of defense responses dependent on BAK1 and SOBIR1 (SUPPRESSOR OF BIR1 1), suggesting that the MC2 prodomain specifically boosts the immunity mediated by the receptor-like protein (RLP)-type PRRs. These data reveal a novel functional and mechanistic link bridging an evolutionarily conserved metacaspase and the regulation of plant PRR signaling. Overall, my thesis work provides valuable insights into the understanding of the complicated regulatory network of PRR signaling in Arabidopsis. These findings can potentially be utilized to engineer durable and broad-spectrum resistance in crops."@en . "https://circle.library.ubc.ca/rest/handle/2429/73244?expand=metadata"@en . "REGULATION OF THE PATTERN-RECOGNITION RECEPTOR SIGNALING IN ARABIDOPSIS: LESSONS FROM SOBIR7-1 AND MC2 by Di Wu B.Sc., Northwest A&F University, 2014 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) January 2020 \u00C2\u00A9 Di Wu, 2020 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: REGULATION OF THE PATTERN-RECOGNITION RECEPTOR SIGNALING IN ARABIDOPSIS: LESSONS FROM SOBIR7-1 AND MC2 submitted by Di Wu in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Botany Examining Committee: Dr. Yuelin Zhang, Botany Supervisor Dr. Jim Kronstad, Plant Science Supervisory Committee Member Dr. Richard Hamelin University Examiner Dr. Geoffrey Wasteneys University Examiner Additional Supervisory Committee Members: Dr. George Haughn, Botany Supervisory Committee Member iii Abstract Plants employ a multi-layered innate immune system to ward off invading pathogens. The pattern-recognition receptors (PRRs) play pivotal roles in immune signaling by recognizing the pathogen-derived conserved molecular signatures termed PAMPs (pathogen-associated molecular patterns). Finetuning the activity of PRR signaling is vital for the well-being of plants, as the perturbation of components of the PRR complex typically causes disastrous outcomes on growth and defense. However, how plants achieve such tight regulation of the PRR signaling is not fully understood. BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), which functions as a coreceptor for PAMP recognition, is an essential component of the PRR complex. BAK1 harbors a mysterious carboxyl-terminal tail (CT) beyond its kinase domain. In the present study, we clarified the biological significance of this CT region using a CT-deletion bak1 mutant allele. Our data showed that the CT is required for BAK1\u00E2\u0080\u0099s function in PAMP-triggered immunity (PTI), but is dispensable for cell death control and brassinosteroid signaling, indicating the differential requirement of the CT in development and immunity. In vitro phosphorylation assay revealed that the CT is required for the autophosphorylation activity of BAK1, suggesting that the CT serves as an intrinsic modulator to promote BAK1\u00E2\u0080\u0099s kinase activity. Plant metacaspases (MCs) are structural homologs of mammalian caspases. Arabidopsis METACASPASE 2 (MC2) carries an N-terminal prodomain, whose function is unclear. Here, we uncovered an imperative role of the MC2 prodomain in activating PTI signaling via genetic analysis of an mc2 mutant allele called mc2-1. Heightened expression of the MC2 prodomain in mc2-1 results in constitutive activation of defense responses dependent on BAK1 and SOBIR1 (SUPPRESSOR OF BIR1 1), suggesting that the MC2 prodomain specifically boosts the immunity iv mediated by the receptor-like protein (RLP)-type PRRs. These data reveal a novel functional and mechanistic link bridging an evolutionarily conserved metacaspase and the regulation of plant PRR signaling. Overall, my thesis work provides valuable insights into the understanding of the complicated regulatory network of PRR signaling in Arabidopsis. These findings can potentially be utilized to engineer durable and broad-spectrum resistance in crops. v Lay Summary Just like humans, plants get sick when they are infested by microbial pathogens. Plant diseases on crops not only cause tremendous yield losses but also threaten food safety. In nature, plants use various defense proteins to fight against pathogen attack, some of which function as sentries, or more formally, receptors. These immune receptors can detect the molecules from pathogens and then activate defense. Since these receptors are very important, plants have evolved multiple strategies to precisely control their activity. In the first project, we found that the tail region of a helper receptor, which helps with activating the real receptors, is important for its function. In the second project, we discovered a new role of a protease in promoting the activity of immune receptors. Interestingly, this protease does not behave as scissors, which they normally do. Overall, my thesis projects expand our understanding of plant defense strategies. vi Preface The studies reported in this Ph.D. thesis covered the research performed from September 2014 to August 2019. Details of the publication and the contribution of the candidate are listed below. Chapter 2 - The carboxyl-terminus (CT) tail of BAK1 is differentially required for its function in development and immunity, is adapted from the following publication: Wu, D., Liu, Y., Xu, F., and Zhang, Y. (2018). \u00E2\u0080\u009CDifferential requirement of BAK1 C-terminal tail in development and immunity.\u00E2\u0080\u009D J Integr Plant Biol 60:270-275. \u00E2\u0080\u00A2 The candidate performed most of the experiments. Xu, F. carried out the genetic crosses of sobir7-1 pad4-1 bir1-1 and helped with some genotyping tasks. The candidate wrote the manuscript and Zhang, Y. revised the manuscript. Chapter 3 - The prodomain of Arabidopsis Metacaspase 2 positively regulates immune signaling mediated by pattern-recognition receptors, was based on the following unpublished manuscript: Wu, D.*, Xu, F.*, Gao, F. and Zhang, Y. \u00E2\u0080\u009CArabidopsis METACASPASE 2 promotes the pattern-recognition receptor signaling via its prodomain.\u00E2\u0080\u009D. In preparation. (*These authors contributed equally to this work) \u00E2\u0080\u00A2 Xu, F. performed genetic crosses of mc2-1 with bak1-4, sobir1-12, bak1-5 bkk1-1, agb1-2 and identified all the desired high-order mutants. Wu, D. helped with genotyping and mutant characterization in the epistasis analysis. Xu, F. initiated the EMS mutagenesis and himself identified all putative suppressor mutants. Wu, D. confirmed the suppressor phenotypes and generated backcross populations for each suppressor line except 6-11-2, vii and 6-15-2, which Xu, F. did. Wu, D. performed positional cloning of all mc2-1 suppressors and carried out mechanistic studies including testing the BAK1/SOBIR1 direct cleavage hypothesis, the \u00E2\u0080\u0098type-II MC inhibitor\u00E2\u0080\u0099 model, and the \u00E2\u0080\u0098dominant-negative effects\u00E2\u0080\u0099 model. Wu, D. characterized the immune phenotypes of mc2-2 and mc2-3 and investigated the physical interaction of MC2 prodomain with CPK28 and BIR1. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables .............................................................................................................................. xiv List of Figures ...............................................................................................................................xv List of Abbreviations ................................................................................................................ xvii Acknowledgements .................................................................................................................. xxiii Dedication ...................................................................................................................................xxv Chapter 1: Introduction ................................................................................................................1 1.1 Overview of the plant immune system ........................................................................... 1 1.1.1 Plant defense mechanisms: from preformed barriers to inducible immunity ............. 1 1.1.2 PAMP-triggered immunity (PTI) ................................................................................ 2 1.1.3 Effector-triggered susceptibility (ETS) ....................................................................... 3 1.1.4 Effector-triggered immunity (ETI) ............................................................................. 4 1.2 Typical immune signaling events downstream of pathogen recognition........................ 7 1.2.1 Activation of mitogen-activated protein (MAP) kinase cascades ............................... 7 1.2.2 PAMP-triggered ROS burst ........................................................................................ 8 1.2.3 Ca2+ signaling in plant immunity .............................................................................. 10 1.2.4 Salicylic acid (SA) biosynthetic and signaling pathways in defense ........................ 10 ix 1.3 The multifaceted function of BAK1 and its homologs in plant development and immunity ................................................................................................................................... 12 1.3.1 BAK1 and BKK1 are important co-receptors for PTI signaling .............................. 13 1.3.2 Specific SERKs are involved in Brassinosteroid (BR) signaling ............................. 13 1.3.3 BAK1 and BKK1 are involved in the negative regulation of cell death .................... 14 1.4 The immune functions of SOBIR1 and BIR1 ............................................................... 15 1.4.1 SOBIR1 is a conserved adapter RLK for RLP-type PRRs ....................................... 15 1.4.2 BIR1 is a negative regulator of the RLP-type PRR complex ................................... 17 1.5 Plant metacaspases ........................................................................................................ 18 1.5.1 The taxonomy of plant proteases .............................................................................. 18 1.5.2 Metazoan apoptotic caspases .................................................................................... 20 1.5.3 C14 family caspase-like proteases ............................................................................ 21 1.5.4 Substrate specificity and calcium dependency of metacaspases ............................... 22 1.5.5 Controversy regarding the functional relevance of metacaspase prodomain ........... 24 1.5.6 Regulatory roles of metacaspases in cell death and beyond ..................................... 25 1.6 Thesis objective ............................................................................................................ 28 Chapter 2: The carboxyl-terminus tail (CT) of BAK1 is differentially required for its function in development and immunity .....................................................................................29 2.1 Summary ....................................................................................................................... 29 2.2 Introduction ................................................................................................................... 29 2.3 Materials and methods .................................................................................................. 31 2.3.1 Growth conditions and mutant generation ................................................................ 31 2.3.2 Plasmid construction ................................................................................................. 32 x 2.3.3 Total protein extraction and western blot ................................................................. 32 2.3.4 Measurement of PAMP-triggered reactive oxygen species (ROS) .......................... 33 2.3.5 PAMP-triggered activation of MAP kinases ............................................................ 33 2.3.6 RNA extraction and quantitative reverse transcription PCR (qPCR) ....................... 33 2.3.7 Measurement of Arabidopsis hypocotyl growth ....................................................... 34 2.3.8 Bimolecular fluorescence complementation (BiFC) assay ....................................... 34 2.3.9 Trypan blue staining ................................................................................................. 34 2.3.10 Purification of His-tagged BAK1 kinase domain from E. coli ............................. 35 2.3.11 Statistical analysis ................................................................................................. 36 2.4 Results ........................................................................................................................... 38 2.4.1 The sobir7-1 mutation causes deletion of the carboxyl-terminal tail of BAK1 ....... 38 2.4.2 BAK1 CT is not required for its function in cell death control ................................ 39 2.4.3 The sobir7-1 and sobir7-1 bkk1-1 mutants are compromised in the PAMP-triggered immunity ............................................................................................................................... 41 2.4.4 BAK1 CT is not essential for brassinosteroid (BR) signaling .................................. 43 2.4.5 Loss of CT does not affect the protein stability of BAK1 ........................................ 45 2.4.6 Deletion of the CT of BAK1 does not compromise its capacity to interact with FLS2 and BIK1 ............................................................................................................................... 45 2.4.7 The CT region is required for the kinase activity of BAK1 ..................................... 48 2.4.8 The CT extension is broadly distributed throughout the LRR-RLK family ............. 50 2.5 Discussion ..................................................................................................................... 52 Chapter 3: The prodomain of Arabidopsis Metacaspase 2 positively regulates immune signaling mediated by pattern-recognition receptors ...............................................................56 xi 3.1 Summary ....................................................................................................................... 56 3.2 Introduction ................................................................................................................... 56 3.3 Materials and methods .................................................................................................. 59 3.3.1 Plant materials and growth condition ....................................................................... 59 3.3.2 Molecular cloning and plasmid construction ............................................................ 60 3.3.3 EMS Mutagenesis and mc2-1 suppressor screen ...................................................... 61 3.3.4 Mapping by sequencing with backcross population ................................................. 61 3.3.5 Pathogen infection assays ......................................................................................... 62 3.3.6 Gene expression analysis .......................................................................................... 62 3.3.7 Firefly luciferase complementation assay ................................................................. 63 3.3.8 Membrane fractionation assay .................................................................................. 63 3.3.9 Co-immunoprecipitation (Co-IP) assay .................................................................... 64 3.4 Results ........................................................................................................................... 67 3.4.1 Metacaspase 2 (MC2) is predominantly expressed in leaf tissue and transcriptionally upregulated upon pathogen infection .................................................................................... 67 3.4.2 MC2 localizes to both plasma membrane and cytosolic space ................................. 70 3.4.3 Epistatic analysis of the mc2-1 autoimmune mutant ................................................ 72 3.4.3.1 Knocking out either BAK1 or BKK1 strongly suppresses the autoimmune phenotype of mc2-1........................................................................................................... 72 3.4.3.2 Loss of SOBIR1 function fully rescues the autoimmunity of mc2-1 ................ 74 3.4.3.3 Results of additional candidate genes in the epistatic analysis ......................... 75 3.4.4 MC2 does not directly cleave BAK1 or BKK1 ........................................................ 76 3.4.5 MC2 is unlikely to regulate immunity via directly cleaving SOBIR1 ...................... 77 xii 3.4.6 Characterization of mc2-1 suppressors ..................................................................... 81 3.4.7 Positional cloning of the rom mutations with mapping-by-sequencing ................... 82 3.4.8 Overexpression of the MC2 prodomain activates immunity .................................... 85 3.4.9 MC2 is unlikely to function by repressing type-II metacaspases ............................. 87 3.4.10 Overexpression of MC1 or MC3 in mc2-1 strongly rescued its autoimmune phenotypes ............................................................................................................................ 90 3.4.11 MC2 is required for basal immunity and nlp20-triggered PTI responses ............. 93 3.4.12 The MC2 prodomain associates with BIR1, but not CPK28, in the co-immunoprecipitation assay ................................................................................................... 95 3.5 Discussion ..................................................................................................................... 96 3.5.1 MC2 prodomain has an unprecedented immune-activating role .............................. 97 3.5.2 Working models illustrating the function of MC2 in plant immunity .................... 100 3.5.2.1 Releasing the repression of BIR1 by the MC2 prodomain ............................. 100 3.5.2.2 Promoting the activity of BAK1/BKK1 or SOBIR1 by MC2 prodomain ...... 101 3.5.3 Overexpression of MC2 prodomain in the mc2-1 mutant likely results from promiscuous enhancer elements inside the T-DNA ............................................................ 103 3.5.4 Potential self-association-mediated functional control of Arabidopsis type-I metacaspases ....................................................................................................................... 103 Chapter 4: Conclusions and future perspectives ....................................................................105 4.1 Conclusions ................................................................................................................. 105 4.2 Future perspectives ..................................................................................................... 107 4.2.1 Regulation of BAK1 function by the CT extension................................................ 107 4.2.2 Regulation of PAMP-triggered immunity by MC2 ................................................ 108 xiii Bibliography ...............................................................................................................................111 xiv List of Tables Table 2.1 Primers used in Chapter 2 ............................................................................................. 36 Table 3.1 Primers used in Chapter 3 ............................................................................................. 64 xv List of Figures Figure 1.1 An overview of the plant immune system. .................................................................... 6 Figure 1.2 Comparison between receptor-like kinase (RLK)-type (left) and receptor-like protein (RLP)-type (right) receptor complex formation. .......................................................................... 17 Figure 1.3 Comparison of the structural organization of different groups of C14 family protease....................................................................................................................................................... 22 Figure 2.1 The sobir7-1 mutation leads to deletion of Carboxyl-terminal tail (CT) of BAK1. ... 39 Figure 2.2 BAK1 CT is dispensable for its function in suppressing spontaneous cell death. ...... 40 Figure 2.3 The sobir7-1 single and sobir7-1 bkk1-1 double mutants are defective in pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI) responses. ............................. 43 Figure 2.4 BAK1 CT is not essential for brassinosteroid (BR) signaling. ................................... 44 Figure 2.5 Loss of CT does not affect the protein stability of BAK1. .......................................... 45 Figure 2.6 Loss of CT of BAK1 does not compromise its ability to interact with FLS2 in split luciferase assay. ............................................................................................................................ 46 Figure 2.7 The SOBIR7-1 mutant protein retains the ability to associate with BIK1. ................. 47 Figure 2.8 The CT of BAK1 promotes its autophosphorylation activity. .................................... 49 Figure 2.9 The CT extensions are widely distributed throughout the leucine-rich repeat receptor-like kinase (LRR-RLK) family. .................................................................................................... 52 Figure 3.1 The phenotype of three metacaspase 2 (mc2) mutant alleles and the expression pattern of MC2. ......................................................................................................................................... 69 Figure 3.2 MC2 localizes to the plasma membrane and cytosolic space...................................... 71 xvi Figure 3.3 Knocking out either BAK1 or BKK1 strongly suppresses the autoimmune phenotypes of mc2-1.\u00CF\u00AE ...................................................................................................................................... 73 Figure 3.4 Loss-of-function mutation of SOBIR1 fully rescues the autoimmune phenotype of mc2-1.\u00CF\u00AE .......................................................................................................................................... 75 Figure 3.5 MC2 does not target BAK1/BKK1 for cleavage. ........................................................ 79 Figure 3.6 MC2 likely does not directly cleave SOBIR1. ............................................................ 80 Figure 3.7 Morphology of seven putative mc2-1 suppressor lines characterized in this study. ... 82 Figure 3.8 The rom1 mutants are intragenic suppressors. ............................................................ 84 Figure 3.9 Elevated expression of MC2 prodomain (PD) leads to autoimmunity........................ 87 Figure 3.10 The selected type-II metacaspases did not physically associate with MC2 and failed to rescue mc2-1 autoimmune phenotypes. .................................................................................... 89 Figure 3.11 Overexpression of MC1 or MC3 largely rescued the autoimmunity of mc2-1. ........ 91 Figure 3.12 MC2 is required for nlp20-induced immune responses and basal immunity. ........... 94 Figure 3.13 The MC2 prodomain physically interacts with BIR1, but not CPK28 in planta. ..... 96 Figure 3.14 Multiple alignment of the prodomain sequence of three Arabidopsis type-I metacaspase by MAFFT (v7.452)................................................................................................. 99 Figure 3.15 Hypothetical models illustrating how MC2 modulates plant immunity. ................ 102 xvii List of Abbreviations 35S A strong promoter derived from the Cauliflower mosaic virus (CaMV); It usually causes ectopic expression of the target gene in plants AA Amino acid ABRC Arabidopsis Biological Resource Center ACT1 Arabidopsis actin 1, AT2G37620 AGB1 Arabidopsis GTP binding protein beta 1 ANOVA Analysis of variance A. thaliana Arabidopsis thaliana ATP Adenosine triphosphate ATR1 Arabidopsis thaliana recognized 1 Avr Avirulent AvrAC An avirulence gene from Xanthomonas campestris pv. Campestris AvrL567 An avirulence gene from flax rust AvrPto An avirulence gene from Pseudomonas syringae pv. Tomato AvrRPM1 Avirulence factor produced by Pseudomonas syringae AvrRpt2 Avirulence factor produced by Pseudomonas syringae BAK1 BRI1-associated kinase1 BiFC Bimolecular fluorescence complementation BIK1 Botrytis-induced kinase1 BIR1 BAK1-interacting receptor-like kinase BLAST Basic Local Alignment Search Tool BKK1 BAK1-like 1 bp Base pair BR Brassinosteroid BRI1 Brassinosteroid-insensitive1 CaMV Cauliflower mosaic virus Cas9 CRISPR-associated protein 9 xviii CBP60g Calmodulin binding protein 60 g CBB Coomassie Brilliant Blue CC Coiled-coil cDNA Complementary DNA CERK1 Chitin elicitor receptor kinase1 Cfu Colony-forming unit CIK3 CLAVATA3 insensitive receptor kinase 3 ChIP-Seq Chromatin immunoprecipitation-sequencing Chr Chromosome CNL CC-NLR (Coiled-coil Nod-like receptor) Co-IP Co-immunoprecipitation Col-0 An Arabidopsis ecotype; it is also referred as wild type throughout this dissertation CPK5 Calcium-dependent protein kinase 5 CPK28 Calcium-dependent protein kinase 28 CRCK3 Calmodulin-binding receptor-like cytoplasmic kinase 3 CRISPR Clustered Regularly Interspaced Short Palindromic Repeats CT Carboxyl-terminus tail DAMP Danger-associated molecular pattern DWF3 DWARF 3 EDS1 Enhanced disease susceptibility 1 EDS5 Enhanced disease susceptibility 5 EF-Tu Bacterial Elongation Factor Tu EFR EF-Tu receptor elf18 An N-acetylated peptide comprising the first 18 amino acids of bacterial elongation factor Tu EMS Ethyl methanesulfonate; a common chemical mutagen ER ERECTA ET Ethylene ETI Effector-triggered immunity xix EV Empty vector, a control vector without foreign gene insertion EXP8 EXPANSIN A8 FLAG An epitope protein tag consisting of a DYKDDDDK sequence flg22 An 22-amino-acid conserved peptide derived bacteria flagellin FLS2 Flagellin-sensitive 2 FRK1 Flg22-induced receptor-like kinase 1 FW Fresh weight gDNA Genomic DNA GFP Green fluorescent protein H.a. Noco2 Hyaloperonospora arabidopsidis Noco2 HA Hemagglutinin; an epitope protein tag composed of YPYDVPDYA sequence His 6\u00C3\u0097His-tag; an epitope tag often used in recombinant protein purification HopM1 An avirulence gene from Pseudomonas syringae pv. Tomato DC3000 HR Hypersensitive response IB Immunoblotting IP Immunoprecipitation ICS1 Isochorismate synthase 1 KD Kinase domain kDa kilo-Dalton LB Lysogeny broth Luc Firefly luciferase LRR Leucine-rich repeat LRR-RLK Receptor-like kinase containing a leucine-rich repeat domain LSD1 Lesion simulating disease 1 MAPK/MPK Mitogen-activated protein kinase MAP2K/MAPKK MAP kinase kinase MAP3K/MAPKKK MAP kinase kinase kinase xx MC Metacaspase MC2 Metacaspase 2 MC2-Z MC2 zymogen MC2CA A catalytic-dead mutant version of MC2 in which the catalytic cysteine is substituted with alanine MC2-PD The genomic DNA sequence coding for the MC2 prodomain MS Murashige and Skoog N. benthamiana Nicotiana benthamiana NLP Necrosis and ethylene-inducing peptide 1-like proteins nlp20 A conserved 20-amino-acid fragment of NLPs NLR NOD-like receptor Nluc/Cluc N-terminus luciferase/C-terminus luciferase NOD Nucleotide binding and oligomerization domain NPR1 Non-expressor of PR genes 1 NPR3 NPR1-like protein 3 NPR4 NPR1-like protein 4 OD Optical density OE Over-expression PCR Polymerase chain reaction PAD4 Phytoalexin deficient 4 PAL Phenylalanine ammonia lyase PAMP Pathogen-associated molecular pattern PBS3 Amido-transferase AvrPphB SUSCEPTIBLE3 PD Pro-domain pep23 An endogenous 23-amino-acid DAMP peptide PR genes Pathogenesis-related genes PRK5 Pollen receptor like kinase 5 PRR Pattern-recognition receptor P.s.m. ES4326 Pseudomonas syringae pv. maculicola ES4326 PTI PAMP-triggered immunity xxi P.s.t. DC3000 Pseudomonas syringae pv tomato DC3000 P.s.t. DC3000 hrc Pseudomonas syringae pv tomato DC3000 hrcC; a mutant strain defective in producing the type three secretion system pv Pathovar qPCR Quantitative PCR R protein Resistance protein RBOHD Respiratory burst oxidase homolog D RIN4 Rpm1-interacting protein 4 RLCK Receptor-like cytoplasmic kinase RLK Receptor-like kinase RLP Receptor-like protein RLP23 Receptor like protein 23 ROS Reactive oxygen species ROT3 ROTUNDIFOLIA 3 RPM1 Resistance to P. syringae pv maculicola 1 RPS2 Resistance to P. syringae 2 RT-PCR Reverse transcriptase PCR SA Salicylic acid SAR Systemic acquired resistance SARD1 SAR deficient 1 SD Standard deviation SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SERK Somatic embryogenesis receptor kinase SIPK SA-induced protein kinase SNC1 SUPPRESSOR OF NPR1, CONSTITUTIVE, 1, a TNL SOBIR1 Suppressor of bir1 1 SOBIR7 Suppressor of bir1 7 SUMM2 Suppressor of MKK1/MKK2 2 T-DNA Transfer-DNA xxii TbMC2 Trypanosoma brucei metacaspase 2 TIR Toll interleukin receptor domain TMK1 Transmembrane kinase 1 TNL TIR-NLR (Toll/Interleukin-1-like nod-like receptors) TTSS Type three secretion system UTR Untranslated region in mRNA WIPK Wound-induced protein kinase WRKY29 WRKY family transcription factor 29 WT Wild type Y2H Yeast two-hybrid YCE C-terminal fragment of the yellow fluorescence protein YFP Yellow fluorescent protein YNE N-terminal fragment of the yellow fluorescence protein xxiii Acknowledgements I am incredibly grateful for having so many wonderful people accompanying me through my Ph.D. journey. The present thesis wouldn\u00E2\u0080\u0099t have been completed without the help of many individuals. First, I would like to express my sincere gratitude to my supervisor, Dr. Yuelin Zhang. An excellent mentor as he, always inspires the students to absorb new knowledge and to seek creative solutions for a given research question. His passion and vision on research are infectious and I consider myself lucky to have the opportunity to learn from him in the past five years. I also offer my great gratitude to my supervisory committees, Dr. George Haughn and Dr. Jim Kronstad, for enlarging my vision of scientific research in their lectures and providing insightful feedback on my research projects in the progress meetings. Their polished lecturing skills have taught me useful lessons on teaching, from which I will benefit substantially in my future career. Some special thanks are owed to Dr. Xin Li for her generous advice and careful reading of my manuscripts. I am also grateful to all peer labmates from Zhang lab and li lab. It was a fun experience working together as a big family. Special thanks are given to Dr. Yukino Nitta, who mentored me at the beginning of my Ph.D. program; to Dr. Yanan Liu, who gave me various useful suggestions on lab work and personal life; to Dr. Tongjun Sun, who offered me tremendous help on many useful lab techniques; to Dr. Jianhua Huang, whose critical questions always promote me to explore scientific questions deeper; to Fan Xu, who helped me transit to the MC2 project smoothly; to Rowan van Wersch, who gave me critical feedback and excellent suggestions on my writing practices. xxiv Funding for this research is provided by the Natural Sciences and Engineering Research Council (NSERC), China Scholarship Council (CSC), and Mitacs Accelerate Fellowship. I sincerely appreciate the financial support from these funding agencies. Thanks to my mother, Nian\u00E2\u0080\u0099e Xiao, who supported me through nearly 20 years of education. Nothing would have been achieved without her love, encouragement, and to a certain degree, sacrifice. Thanks to my brother, Long Wu, who accompanies me along the way and constantly encourages me. I also want to thank many friends; festival potlucks, Friday night drinking, and weekend skiing trips always keep me reenergized whenever exhausting bench work pushes me to the verge of mental breaking down. And finally, thanks to Siyu Song, for her love, support, and company. xxv Dedication Dedicated to my mother, Nian\u00E2\u0080\u0099e Xiao, who gives me life and endless love. 1 Chapter 1: Introduction 1.1 Overview of the plant immune system 1.1.1 Plant defense mechanisms: from preformed barriers to inducible immunity Plants are undoubtedly essential for the well-being of humans. They are literally the foundation of the Earth\u00E2\u0080\u0099s ecosystem upon which every aspect of human life is built. Plants provide us with not only food resources but also with various essential non-food products such as wood, medicines, and fuel. In order to feed the surging human population, sustainable and high-yield crop cultivation is one of the top priorities of our time. However, plants in a natural habitat often suffer from severe diseases caused by various pests such as pathogenic microbes. Estimates of global yield losses due to plant disease range up to 20-40 percent, amounting to approximately 290 billion dollars per year (Savary et al., 2012). To alleviate such dreadful losses, it is vital to understand the molecular mechanisms that underlie the formation of plant disease resistance. Plants are not just helpless victims. Despite lacking adaptive immunity as possessed by mammals, plants have evolved an elaborate innate immune system to fight against pathogen attack. The frontline of this immune system consists of preformed physical barriers such as a waxy epidermal cuticle and rigid plant cell walls, both serving as fortifications to block pathogen entry, along with chemical barriers, which include diverse surface-deposited anti-microbial compounds (Hamuel, 2015). Once pathogens have access to plant interiors, for example, by stealthily entering from stomata or forcefully penetrating across the cuticle, plant cells can detect their presence, and upon detection mount rapid and robust immune responses. Such inducible immune responses typically involve strengthening of physical barriers, secretion of antimicrobial compounds, synthesis of immune phytohormones, production of pathogenesis-related (PR) proteins, and 2 activation of programmed cell death (PCD). Depending on the identity of the stimuli, this defense system is divided into two conceptual layers, namely PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) (Figure 1.1) (Chisholm et al., 2006; Jones and Dangl, 2006). 1.1.2 PAMP-triggered immunity (PTI) PAMP-triggered immunity (PTI) is activated immediately following recognition of pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) by the plasma membrane-localized pattern-recognition receptors (PRRs). Such PAMPs are typically the signature molecules essential for a pathogen\u00E2\u0080\u0099s lifestyle. Notable PAMP representatives are bacteria flagellin which is the building block of bacterial flagella; prokaryotic translation factor protein EF-Tu; and fungal chitin, the structural polysaccharide of the fungal cell wall. Moreover, endogenous stimuli, termed damage-associated molecular patterns (DAMPs), can also trigger immunity and are perceived by plant PRRs in a manner analogous to pathogen-derived PAMPs. Extensive studies on PAMP perception have revealed the cognate host PRR receptors for many PAMPs. Typical plant PRRs belong to the receptor-like kinase (RLK) family or the receptor-like protein (RLP) family. Structurally speaking, RLKs share similar architectural composition with metazoan tyrosine receptor kinases (RTKs), consisting of a unique extracellular domain, a transmembrane domain, and an intracellular kinase domain, whereas RLPs lack an intracellular domain. Plant genomes typically encode a vast array of RLKs and RLPs, with ~410 RLKs and ~170 RLPs annotated in the model plant Arabidopsis thaliana (hereafter A. thaliana or Arabidopsis), and ~917 putative RLKs and ~90 RLPs in rice (Fritz-Laylin et al., 2005; Li et al., 2016; Shiu et al., 2004). Perhaps the most famous PRR in Arabidopsis is the RLK FLS2 (FLAGELLIN SENSING 2), which detects flagellin or its elicitor-active epitope flg22 (Chinchilla 3 et al., 2006; Gomez-Gomez and Boller, 2000). Some other well-characterized PAMP/DAMP-PRR pairs include EF-Tu-EFR (Zipfel et al., 2006a), NLPs (necrosis- and ethylene-inducing protein 1-like proteins)-RLP23 (Albert et al., 2015), chitin-CERK1 and pep1-PEPR1/2 (Yamaguchi et al., 2010; Yamaguchi et al., 2006). Recognition of PAMPs at the cell surface triggers a series of downstream defense responses, including activation of mitogen-activated protein (MAP) kinase cascades, generation of reactive oxygen species (ROS), accumulation of defense hormones such as salicylic acid (SA) and ethylene, secretion of antimicrobial phytoalexins, and extensive transcriptional reprogramming of defense-related genes (Figure 1.1) (Peng et al., 2018). Collectively, these PTI responses contribute to the restriction of pathogen entry and proliferation by creating an adverse environment for pathogen growth as well as minimizing nutrient leakage. 1.1.3 Effector-triggered susceptibility (ETS) Well-adapted pathogens manage to suppress PTI responses and regain dominance in the battle against their plant hosts. This process is termed effector-triggered susceptibility (ETS) (Chisholm et al., 2006; Jones and Dangl, 2006). Pathogens typically achieve ETS through delivering effector proteins to the apoplast and into host cells, where these effectors interfere with PTI signaling pathways and manipulate the host metabolism in favor of pathogen fitness (Chisholm et al., 2006; Jones and Dangl, 2006). Despite substantial diversity in the structure and enzymatic activity of effectors, pathogens\u00E2\u0080\u0099 virulence strategies tend to converge upon targeting the central regulatory hubs or pivotal signaling nodes in the immune system (Mukhtar et al., 2011; Wessling et al., 2014). For example, pathogens often target the components involved in the biosynthesis and signaling transduction steps of salicylic acid (SA), an essential defense hormone. 4 Cmu1, a chorismate mutase secreted from the fungus U. maydis, dampens the accumulation of SA by redirecting its precursor into other unrelated biosynthetic routes (Djamei et al., 2011). Likewise, two effectors from the oomycete P. sojae and the fungus V.dahliae, PsIsc1 and VdIsc1, respectively, convert SA precursor isochorismate into 2,3-dihydro-2,3-dihydroxybenzoate and pyruvate, thereby hindering SA accumulation (Liu et al., 2014). Moreover, some effectors promote virulence through facilitating nutrient transfer such as two transcription activator-like (TAL) effectors PthXo1 and AvrXa7 from Xanthomonas oryzae (Yang et al., 2006; Yang et al., 2000), or through creating an aqueous apoplast environment favorable for infection, best exemplified by HopM1 from Pseudomonas syringae (Xin et al., 2016). 1.1.4 Effector-triggered immunity (ETI) To prevail in the evolutionary arms race against pathogens, plants have evolved a second layer of immunity, termed effector-triggered immunity (ETI), to counteract the activity of effectors (Chisholm et al., 2006; Jones and Dangl, 2006). ETI is generally a more robust defense response and is often accompanied by localized programmed cell death known as the hypersensitive response (HR). ETI is triggered upon recognition of effectors by plant resistance proteins (R proteins). The majority of R proteins belong to the intracellular Nod-like receptor (NLR) protein family. Structurally speaking, canonical NLRs contain three functional domains: an N-terminal Toll/interleukin-1 receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding and oligomerization domain (NOD), and a C-terminal leucine-rich repeat (LRR) domain. Due to their N terminus difference, canonical NLRs are classified into TIR- and CC-types, designated as TNL and CNL, respectively. 5 NLRs recognize their cognate effectors either directly by physical binding or indirectly, via guarding the integrity of host proteins targeted by effectors. So far, examples of direct recognition remain limited. For instance, in yeast two-hybrid (Y2H) assay, the flax TNLs L5/L6/L7 and M interact with the Melampsora lini effectors AvrL567 and AvrM, respectively (Catanzariti et al., 2010; Dodds et al., 2006). The rice CNL Pi-ta protein directly binds to the Magnaporthe grisea effector AvrPita both in Y2H and in vitro (Jia et al., 2000). In co-immunoprecipitation (Co-IP) assay, Arabidopsis TNL RPP1 specifically associates with oomycete pathogen Hyaloperonospora arabidopsidis (H.a.) effector ATR1 in vivo through its C-terminal LRR domain (Krasileva et al., 2010). Despite strong initial support for the direct recognition model, accumulating evidence demonstrates that the indirect recognition strategy is, in fact, a dominant mechanism for effector detection. Indirect recognition is executed by NLRs that recognize the effector-mediated modifications on the host target proteins termed as guardees, which themselves modulate immunity, or decoys, which are mere structural mimics of genuine immune regulators (Cesari, 2018; van der Hoorn and Kamoun, 2008). Such an indirect mechanism is likely to allow for a broader pathogen detection spectrum given a limited repertoire of NLRs in plant genomes (~150 in Arabidopsis and ~460 in rice) (Cesari, 2018). Perhaps the best demonstration of the indirect recognition mechanism is how CNLs RPM1 and RPS2 detect effectors AvrRPM1 and AvrRPS2, respectively, through monitoring the guardee protein named RPM1 interacting protein 4 (RIN4) (Day et al., 2005; Mackey et al., 2002). RPM1 senses the phosphorylation of RIN4 induced by AvrRPM1 and triggers defense (Chung et al., 2011; Liu et al., 2011). AvrRPS2 cleaves RIN4 with its cysteine protease activity and the disappearance of RIN4 triggers RPS2-dependent ETI (Mackey et al., 2002). Another well-studied example is the detection of a P. syringae effector 6 HopAI1 by the CNL SUMM2. HopAI1 harbors phosphothreonine lyase activity and can disrupt the kinase activity of several MAP kinases including MPK4 (Zhang et al., 2012). This disruption of MPK4 kinase activity can be sensed by SUMM2 through diagnosing the abnormal phosphorylation status of an MPK4 substrate protein CRCK3 (Zhang et al., 2017; Zhang et al., 2012). Remarkably, the decoy module can sometimes be directly integrated into the NLR receptors as an effector-sensing domain, as demonstrated by Arabidopsis TNL pair RRS1-RPS4 (Sarris et al., 2015), and rice CNL pair RGA5-RGA4 (Cesari et al., 2014). PAMP, pathogen-associated molecular pattern; PRR, pattern-recognition receptor; PM, plasma membrane; NLR, Nod-like receptor; BAK1, BRI1-ASSOCIATED RECEPTOR KINASE 1; MAPK, Mitogen-activated protein kinase; ROS, reactive oxygen species. SA, salicylic acid; PTI, PAMP-triggered immunity; ETI, effector-triggered immunity. Figure 1.1 An overview of the plant immune system. 7 1.2 Typical immune signaling events downstream of pathogen recognition 1.2.1 Activation of mitogen-activated protein (MAP) kinase cascades MAPK cascades are evolutionarily conserved signaling modules in eukaryotes, responsible for relaying and amplifying signals from membrane-resident receptors to the downstream signaling executors (Meng and Zhang, 2013; Rodriguez et al., 2010b). A typical MAP kinase cascade is composed of three tiers of kinases organized in a hierarchical manner, namely a MAP Kinase Kinase Kinase (MAPKKK/MEKK), a MAP Kinase Kinase (MAPKK/MKK), and a MAP Kinase (MAPK/MPK) (Meng and Zhang, 2013). These components are widely conserved across plant lineages and are often present with multiple copies in plant genomes. Analysis of the Arabidopsis genome reveals 20 MPKs, 10 MKKs and approximately 60 putative MAPKKKs (Meng and Zhang, 2013). In response to stimuli, MEKKs become activated by upstream signaling components. Upon activation, MEKKs phosphorylate downstream MKKs, which in turn phosphorylate and thus activate their cognate MPKs. Activated MPKs then phosphorylate their downstream substrates such as transcription factors or metabolic enzymes to modulate their biochemical function (Meng and Zhang, 2013; Rodriguez et al., 2010b). MAP kinase cascades play important roles in various aspects of plant biology, ranging from plant growth to abiotic stress acclimation to defense responses against pathogens (Rodriguez et al., 2010a). In A. thaliana, two well-characterized MAP kinase cascades are crucial for immune signaling. The first cascade, consisting of MAPKKK3/5-MKK4/5-MPK3/6, is activated by many PAMPs/DAMPs such as flg22, nlp20, and pep1, and positively regulates PTI responses via promoting stomatal immunity, phytoalexin and indole glucosinolate biosynthesis, and ethylene production (Bi et al., 2018; Devendrakumar et al., 2018; Sun et al., 2018). This MAP kinase cascade also contributes to PAMP-induced transcriptional upregulation of diverse defense-related 8 genes, such as FLG22-INDUCED RECEPTOR-LIKE KINASE 1 (FRK1) and WRKY DNA-binding protein 29 (WRKY29) (Asai et al., 2002). WRKY29 and FRK1 have been widely used as molecular markers to assess the PTI magnitude. Besides PTI, MPK3 and MPK6 are also activated in ETI (Tsuda et al., 2013; Zhang and Klessig, 1998). Genetic studies have revealed that the Arabidopsis MPK3/6 and their tobacco orthologs are important for the establishment of NLR-mediated defense and hypersensitive response (Liu et al., 2007; Su et al., 2018). Another well-studied defense-related MAP kinase cascade is composed of MEKK1-MKK1/MKK2-MPK4, which, intriguingly, is involved in both positive and negative regulation of immunity (Devendrakumar et al., 2018). On one hand, this cascade contributes to PTI responses, based on transcriptomics studies showing that MPK4 is required for upregulation of approximately 50% of flg22-induced genes (Frey et al., 2014). In addition, MPK4 can activate camalexin biosynthesis through its substrates MKS1 and WRKY33 (Qiu et al., 2008). On the other hand, this cascade has evolved into a surveillance mechanism to counteract pathogen effectors that target components of the MAP kinase cascades. As mentioned above, the integrity of this cascade is guarded by an NLR protein SUMM2, since the disappearance of any component of this cascade results in activation of SUMM2-mediated ETI responses (Zhang et al., 2017; Zhang et al., 2012). 1.2.2 PAMP-triggered ROS burst Apoplastic ROS burst is one of the earliest PTI outputs. PAMP-induced ROS is initially produced in the apoplastic space as a form of superoxide (O2-) and then converted into the membrane-permeable hydrogen peroxide (H2O2) (Qi et al., 2017). Typically, PAMP-triggered ROS generation emerges at 2-3 min following PAMP treatment and peaks around 10-14 min (Bigeard et al., 2015). In Arabidopsis, PAMP-induced ROS production relies on a plasma 9 membrane-localized NADPH oxidase, named respiratory burst oxidase homolog D (RBOHD) (Kadota et al., 2014; Li et al., 2014). ROS molecules contribute to plant defense through several mechanisms. Due to their high reactivity, ROS are believed to damage pathogens directly. ROS also contribute to the strengthening of host cell walls via cross-linking of the cell wall glycoproteins (Bradley et al., 1992). Furthermore, ROS burst is required for stomatal closure in response to pathogen attack (Li et al., 2014). There is also ample evidence supporting ROS serving as signal molecules through their interplay with other immune signals such as calcium (Ca2+) and reactive nitrogen species (RNS) (Gilroy et al., 2014; Lehmann et al., 2015; Ranf et al., 2011). RBOHD is the key enzyme responsible for PAMP-induced ROS generation (Kadota et al., 2015). The activation of RBOHD is a rate-limiting step of ROS burst and this step is under intricate regulation to optimize the timing and magnitude of ROS synthesis (Kadota et al., 2015; Qi et al., 2017). Mechanistic studies demonstrate that RBOHD activity is precisely controlled by the cooperative action between calcium signaling components including calcium-dependent protein kinases (CDPKs, or CPKs), and receptor-like cytoplasmic kinases (RLCKs) such as BOTRYTIS-INDUCED KINASE 1 (BIK1) (Kadota et al., 2015). RBOHD itself can directly bind to Ca2+ via its N-terminal EF-hand motif and four Ca2+-activated CPKs, CPK4/5/6/11, can phosphorylate RBOHD in vivo to facilitate ROS generation (Dubiella et al., 2013; Kadota et al., 2015). In addition, BIK1 can also phosphorylate RBOHD to promote its activation. The full enzyme activity of RBOHD requires both phosphorylation events from CPKs and RLCKs, highlighting their synergistic contribution in the ROS pathway (Li et al., 2014). 10 1.2.3 Ca2+ signaling in plant immunity PAMP/DAMP recognition triggers a rapid elevation of cytosolic Ca2+ level, which is mediated predominantly by membrane-localized Ca2+ channels including the CNGC2-CNGC4 complex (Seybold et al., 2014; Tian et al., 2019). Specific PAMP/DAMP stimulus generates a unique Ca2+ signature differing in magnitude, duration, and oscillation pattern, which will subsequently be decoded by various Ca2+ sensor proteins, including calcineurin B-like (CBL)-CIPKs kinase complex, CBP60g family transcription factors, and the aforementioned CPKs (Boudsocq et al., 2010; Ranf et al., 2011; Seybold et al., 2014). These Ca2+-activated immune regulators then orchestrate downstream defense responses, such as ROBHD-mediated ROS production, synthesis of phytohormone SA, and transcriptional reprogramming of diverse defense-related genes (Boudsocq et al., 2010; Seybold et al., 2014). 1.2.4 Salicylic acid (SA) biosynthetic and signaling pathways in defense Salicylic acid (SA) has long been recognized as a pivotal defense hormone essential for both basal resistance against (hemi-)biotrophs and the systemic acquired resistance (SAR) (Zhang and Li, 2019). In the model organism A. thaliana, SA biosynthetic pathways and the downstream signaling components have been well dissected. In Arabidopsis, there are two SA biosynthetic pathways reported, termed the ICS (Isochorismate Synthase) pathway and PAL (PHENYLALANINE AMMONIA LYASE) pathway. Among them, the ICS pathway is the predominant source of pathogen-induced SA biosynthesis (Zhang and Li, 2019). In the ICS pathway, the primary precursor chorismate is first converted into isochorismate in the chloroplast through an enzyme called ICS1 (Isochorismate Synthase 1), after which isochorismate is transported into the cytosol before being converted into 11 SA via PBS3 (amido-transferase AvrPphB SUSCEPTIBLE3) (Rekhter et al., 2019; Tian et al., 2019; Zhang and Li, 2019). Mutants defective in the ICS pathway all exhibit severely compromised SAR and basal resistance, confirming the major contribution of this pathway in defense. Regarding the transcriptional regulation of the ICS pathway, expression of the rate-limiting enzyme ICS1 is governed by two central transcription factors, SARD1 (SAR DEFICIENT 1) and CBP60g (calmodulin-binding protein 60-like.g), as genetically supported by abolished pathogen-induced ICS1 expression in the sard1 cbp60g double mutant (Zhang et al., 2010). SARD1 and CBP60g can both bind to the ICS1 promoter region in a chromatin immunoprecipitation (ChIP) assay, confirming that they are the immediate upstream transcriptional activators of ICS1 (Zhang et al., 2010). Signaling components downstream of SA have also been well-characterized. Free SA is initially perceived by SA receptors NPR1 (NONEXPRESSER OF PR GENES 1) and its two paralogues, NPR3 and NPR4 (Cao et al., 1997; Ding et al., 2018; Wu et al., 2012). NPR1 functions as a transcriptional activator, whereas NPR3 and NPR4 act as redundant transcriptional repressors. Binding to SA leads to a conformational change in NPR1, thus unleashing its transcriptional activator capacity (Wu et al., 2012). Conversely, SA binding to NPR3 and NPR4 abrogates their transcription repressor activity, thereby turning on the expression of their target genes (Ding et al., 2018). Both NPR1 and NPR3/4 can associate with redundant TGA transcriptional factors TGA2/5/6 to modulate the expression of defense-related genes such as SARD1, WRKY70, and PR genes, ultimately leading to disease resistance (Ding et al., 2018). 12 1.3 The multifaceted function of BAK1 and its homologs in plant development and immunity BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1, also named SERK3), is an evolutionarily conserved leucine-rich repeat (LRR)-type receptor-like kinase (LRR-RLK). BAK1 belongs to the so-called SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) family with altogether five members (Ma et al., 2016b). The SERKs are structurally and functionally highly-conserved throughout the plant kingdom. SERKs are multi-functional proteins; they control diverse developmental and immune pathways by serving as co-receptors for different surface-localized receptors (Ma et al., 2016b). For example, BAK1 and SERK4 (also named BKK1) coordinate with multiple immune receptors to perceive their matching PAMPs (He et al., 2018). SERK1, BAK1, and BKK1 form a ligand-dependent receptor complex with BRASSINOSTEROID INSENSITIVE 1 (BRI1) to perceive key growth hormone Brassinosteroids (BRs) (Gou et al., 2012; Li et al., 2002b). Moreover, specific SERKs work with ERECTA (ER) family receptors to recognize EPIDERMAL PATTERNING FACTORS (EPFs) for cell fate specification during stomatal patterning (Meng et al., 2015), with PHYTOSULFOKINE RECEPTOR 1 (PSKR1) to detect PHYTOSULFOKINE (PSK) for root growth promotion (Wang et al., 2015), and with the RLK EXCESS MICROSPOROCYTES 1 (EMS1) to sense TAPETUM DETERMINANT 1 (TPD1) for male gametophyte development (Zhao et al., 2002). It has become a common theme that SERK proteins function as universal co-receptors for a wide array of defense and growth receptors, thereby serving as a convergent signaling node that connects various immune and developmental pathways. 13 1.3.1 BAK1 and BKK1 are important co-receptors for PTI signaling In PTI, many PRRs, including FLS2, EFR, PEPR1/2, and RLP23, recruit BAK1 as a co-receptor to form a ligand-dependent heteromeric complex competent for ligand sensing and signaling activation (Couto and Zipfel, 2016; He et al., 2018). Among them, FLS2 is the best-characterized one and could serve as an illustrative paradigm for other BAK1-dependent PRRs (Figure 1.2, left). Upon binding to flg22, FLS2 rapidly recruits BAK1 to assemble into a bipartite receptor complex (Zhao et al., 2002). After trans-phosphorylation, the FLS2-BAK1 complex becomes activated and then phosphorylates downstream components such as RLCKs leading to the activation of PTI (He et al., 2018; Lu et al., 2010b). The recruitment of BAK1 is essential for defense as bak1 knockout mutants show strongly compromised flg22-induced PTI hallmarks (Chinchilla et al., 2007). Crystal structure analysis reveals that the FLS2-BAK1 heterodimer is stabilized by the ligand flg22 which acts as a \u00E2\u0080\u0098molecular glue\u00E2\u0080\u0099, with its C-terminus clenching onto the ectodomain of BAK1 and FLS2 simultaneously (Sun et al., 2013b). The closest homolog of BAK1, BKK1 (also known as SERK4), functions redundantly with BAK1 in the defense signaling, as supported by the more pronounced PTI defects in the bak1 bkk1 double mutant compared to the bak1 single mutant (Roux et al., 2011b). On the other hand, overaccumulation of BAK1 or BKK1 results in severe autoimmune phenotypes such as retarded growth and constitutive expression of PR genes, highlighting the positive roles of these co-receptors in the defense signaling (Domnguez-Ferreras et al., 2015). 1.3.2 Specific SERKs are involved in Brassinosteroid (BR) signaling Brassinosteroids (BRs) are growth-promoting hormones that regulate many aspects of plant growth and development, including cell elongation, photomorphogenesis, root growth, 14 flowering time and male fertility (Zhu et al., 2013). The BR perception and signal transduction pathways have been dissected at length. Briefly, BR binds to the extracellular domain of the surface-localized RLK BRI1, which in turn facilitates a cascade of downstream signaling events, sequentially taking place as follows: phosphorylation of members of the BSK family of kinases and the BSU family of phosphatases; BSU1-mediated dephosphorylation and inactivation of GSK3-like kinases such as BIN2; activation of the BZR1 family transcription factors; and ultimately leading to the transcriptional reprogramming of numerous BR-responsive genes (Zhu et al., 2013). Activation of BRI1 involves the recruitment of the co-receptor BAK1 and the trans-phosphorylation events between the two. However, the BAK1 null mutant, bak1-4, only shows very mild BR signaling defects compared to the receptor mutant bri1, suggesting potential functional redundancy among SERK members (Li et al., 2002b). Indeed, besides BAK1, SERK1 and BKK1 also contribute to BR perception, and the serk1 bak1 bkk1 triple mutant exhibits nearly complete loss of BR sensitivity (Gou et al., 2012). Whereas SERK2 appears to be exempted from the task of BR perception, a recent report revealed that the SERK5 orthologue in the ecotype Landsberg erecta (Ler-0) can interact with BRI1 and has a profound effect on BR responses (Wu et al., 2015). 1.3.3 BAK1 and BKK1 are involved in the negative regulation of cell death Besides its critical roles in plant development and immunity, BAK1 is also involved in the negative regulation of cell death. The bak1 knockout mutant displays spreading cell death and lesions upon infection, as well as premature senescence (Kemmerling et al., 2007). BKK1 plays a redundant role in this cell death inhibition process as the bak1 bkk1 doubly null mutant, but not the respective single mutants, exhibits spontaneous cell death and seedling lethality (He et al., 15 2007). Intriguingly, this cell death phenotype is accompanied by constitutive activation of defense, as evidenced by highly elevated expression of defense marker genes PR1 and PR2 in bak1 bkk1 (He et al., 2007). Recent genetic studies have greatly advanced our understanding of the molecular basis of the death control function of BAK1/BKK1. It is shown that the cell death phenotype of bak1 bkk1 is light-dependent, kinase activity-dependent, and partially SA-dependent (He et al., 2008; Kemmerling et al., 2007). Major breakthroughs arose from recent suppressor screens of the bak1 bkk1 double mutant. Some of those suppressor genes were mapped to pathways involved in N-glycosylation modification, nucleocytoplasmic trafficking, and endoplasmic reticulum (ER)-mediated protein quality control, suggesting the requirement of these pathways in BAK1/BKK1-regulated cell death (de Oliveira et al., 2016; Du et al., 2016). Recently, Yu et al. (2019) demonstrate that a calcium channel-forming protein, CYCLIC NUCLEOTIDE GATED CHANNEL 20 (CNGC20), is required for the cell death and autoimmune phenotypes of bak1 bkk1, highlighting the involvement of calcium signaling in BAK1/BKK1-mediated cell death control. 1.4 The immune functions of SOBIR1 and BIR1 1.4.1 SOBIR1 is a conserved adapter RLK for RLP-type PRRs Plant RLP-type PRRs contain a ligand-binding extracellular domain, and a transmembrane domain but, in sharp contrast to RLK-type PRRs, lack any obvious cytoplasmic domain, suggesting that they require certain signaling partners in order to relay PAMP signals at the cytoplasmic side. A highly-conserved RLK, SUPPRESSOR OF BIR1-1 (SOBIR1), is such a common signaling partner (Liebrand et al., 2014a). SOBIR1 was originally identified in 16 Arabidopsis as a suppressor of the autoimmune mutant bir1-1. To date, the sequence and function of SOBIR1 have been shown to be broadly conserved across diverse plant lineages (Liebrand et al., 2014a). Mechanistically speaking, SOBIR1 constantly associates with its RLP partner to form a stable heterodimeric receptor module, which is functionally equivalent to an RLK-type PRR (Liebrand et al., 2014a). Partners of SOBIR1 in Arabidopsis include the aforementioned NLP receptor RLP23, and RLP30, which detects a proteinaceous elicitor called SsE1 from Sclerotinia sclerotiorum (Albert et al., 2015; Zhang et al., 2013). In tomato (Solanum lycopersicum, Sl), the SlSOBIR1 works with Cf4 and Cf9, two famous RLPs conferring resistance to the fungal pathogen C. fulvum, to recognize their corresponding effectors (Jones et al., 1994; Liebrand et al., 2013; Rivas and Thomas, 2005). Loss of SlSOBIR1 function dampens tomato resistance against C. fulvum, highlighting a critical role of SlSOBIR1 in the basal immunity (Liebrand et al., 2013). The mode of action of the Arabidopsis RLP23 illustrates an elegant paradigm of how RLPs maneuver to detect their matching ligands (Figure 1.2, right). Membrane-resident RLP23 forms a constitutive, ligand-dependent signaling module with SOBIR1 (Albert et al., 2015). Upon binding to its ligand NLPs, or the elicitor-active peptide nlp20, with its unique extracellular leucine-rich repeat domain, RLP23 recruits the coreceptor BAK1 and they instantly assemble into a tripartite receptor complex (Albert et al., 2015), which is functionally equivalent to the aforementioned 17 RLK-BAK1 bipartite complex. This ligand-dependent tripartite complex will then switch on the downstream signaling events (Figure 1.2, right) (Albert et al., 2015). FLS2, FLAGELLIN-SENSITIVE 2; BAK1, BRI1-ASSOCIATED RECEPTOR KINASE 1; BKK1, BAK1-LIKE 1; RLP23, RECEPTOR LIKE PROTEIN 23; SOBIR1, SUPPRESSOR OF BIR1 1. Asterisks represent the corresponding ligands of the immune receptors. 1.4.2 BIR1 is a negative regulator of the RLP-type PRR complex As its name suggested, SOBIR1 was originally identified as a suppressor of an autoimmune mutant bir1-1, which contains a loss-of-function mutation in BAK1-INTERACTING RECEPTOR-LIKE KINASE 1 (BIR1). Other suppressor genes of bir1-1 include AGB1 (GTP BINDING PROTEIN BETA 1) and BAK1 (Liu et al., 2013b; Liu et al., 2016). To date, the mechanistic explanation of bir1-1 autoimmunity has been unraveled. BIR1 is a regulatory RLK that constantly associates with BAK1 to block its interaction with the SOBIR1-RLPs receptor module. The Figure 1.2 Comparison between receptor-like kinase (RLK)-type (left) and receptor-like protein (RLP)-type (right) receptor complex formation. 18 absence of BIR1 leads to promiscuous complex formation between BAK1 and SOBIR1-RLPs, explaining why the null mutant bir1-1 displays SOBIR1- and BAK1-dependent autoimmune phenotypes (Gao et al., 2009; Liu et al., 2016). The catastrophic bir1-1 autoimmune phenotypes highlight the necessity of negative regulation on PRR-mediated immunity. A handful of such negative regulators have been uncovered from previous genetic screens (Couto and Zipfel, 2016). For example, an active PP2A phosphatase inhibits the function of the PRR complex by dephosphorylating BAK1 (Segonzac et al., 2014). CPK28 attenuates PTI signaling by promoting the protein turnover of the RLCK BIK1 (Monaghan et al., 2014). NIK1, an RLK, associates with FLS2 and BAK1, thus negatively impacting the formation of the FLS2/BAK1 complex (Li et al., 2019). More of such negative regulators await discovery in future research. 1.5 Plant metacaspases 1.5.1 The taxonomy of plant proteases Proteolysis is a process of protein catabolism. It is conducted by cellular enzymes termed as proteases or peptidases, which hydrolyze peptide bonds. Proteases play profound roles in many aspects of plant biology. A fundamental role of protease is to degrade non-functional proteins into amino acids for recycling. However, the impact of proteases is far beyond such a basic housekeeping metabolic role. In fact, by irreversibly controlling the fate of the substrate proteins through cleavage, they serve as pivotal regulators in a diverse array of developmental and physiological processes. Such regulatory proteases normally exhibit strong substrate specificity, usually only cleaving a group of proteins sharing particular motifs; and oftentimes, the P1 position, which is the residue immediately toward the N-terminus of the scissile peptide bond, is a 19 predominant substrate determinant (van der Hoorn, 2008). Notably, the outcome of proteolysis could also vary depending on the nature of substrates. Whereas the majority of substrates are destroyed due to cleavage, certain proteins can be activated through proteolysis via mechanisms such as removing certain inhibitory domains or releasing the actual bioactive truncated fragment. Biochemically speaking, the first step of proteolysis is the polarization of the carbonyl group of the target peptide bond, which is achieved by stabilizing the oxygen atom in an oxyanion hole constructed by the active protease (van der Hoorn, 2008). Afterward, an activated nucleophile attacks the carbon atom of the polarized carbonyl group, leading to the breakage of the peptide bond. Depending on the donor residue of the nucleophile, proteases are categorized into four catalytic classes: cysteine proteases, serine proteases, metalloproteases, and aspartic proteases. On the basis of the evolutionary relationships, the MEROPS protease database further divides these four classes of proteases into different clans and even further into families (Rawlings et al., 2018; Rawlings et al., 2010). For example, the Arabidopsis genome encodes more than 800 putative proteases, spreading across nearly 60 families of 30 different clans (van der Hoorn, 2008). Cysteine proteases use a catalytic cysteine as the nucleophile when catalyzing proteolysis. The C14 family, also known as the caspase family, belongs to the CD clan of cysteine protease, featuring a conserved Cys-His catalytic dyad and a unique tertiary structure named caspase-hemoglobinase fold (Klemencic and Funk, 2019). The best-known representatives of C14 family are the metazoan caspases (cysteine-dependent aspartate-directed proteases), due to their crucial roles in animal apoptosis as well as in inflammatory responses. 20 1.5.2 Metazoan apoptotic caspases Metazoan caspases are highly specific proteases that recognize particular tetrapeptide motifs on the substrate and perform cleavage after an Asp residue. Based on the structural differences, canonical apoptotic caspases are classified into two groups: initiator caspases and executioner caspases. Structurally, initiator caspases such as human caspase 8 are composed of an N-terminal segment termed prodomain, which oftentimes is a caspase recruitment domain (CARD) or death effector domain (DED), and a C-terminal catalytic domain formed by two conserved subunits named p20 and p10 (MacKenzie and Clark, 2012). The executioner caspases such as human caspase 3 lack a prodomain and have only the core peptidase domain (MacKenzie and Clark, 2012). The apoptotic pathways in animals have been extensively characterized. Caspases are first synthesized in an inactive zymogen form. Recognition of extrinsic or intrinsic death signals results in oligomerization and self-processing of initiator caspases, leading to their activation (Nagata, 2018). Notably, the N-terminal prodomains of initiator caspases mediate the self-association and are normally removed through auto-processing (MacKenzie and Clark, 2012). Active initiator caspases then cleave executioner zymogens at the linker region between the p20 and p10 subunits, thereby unleashing their protease activity. Activated executioner caspases dimerize via the p10 subunit and then cleave a plethora of substrates including proteins involved in fundamental metabolism and cytoskeleton dynamics, ultimately leading to the collapse of the cellular structure and cell death (Nagata, 2018). 21 1.5.3 C14 family caspase-like proteases Apart from caspases, the C14 family also contains another three other groups of proteases: orthocaspases, paracaspases, and metacaspases (MCs) (Uren et al., 2000). These proteases are collectively termed as caspase-like proteases because they all share the signature p20 subunit, which carries the Cys-His catalytic dyad embedded in the characteristic caspase-haemoglobinase fold. Caspase-like proteases are present in virtually all forms of life, including protozoa, fungi, plants, and even prokaryotes. Despite the high conservation of the p20 subunit, the overall protein structure and global sequence similarity vary substantially across distinct groups of the C14 family proteases (Figure 1.3). Orthocaspases, the prokaryotic p20-containing counterparts in bacteria, are devoid of a p10 subunit but sometimes carry an extra domain positioned after the p20 catalytic domain. Similarly lacking a p10 subunit, paracaspases, which are exclusively found in the genomes of slime molds and metazoan organisms, often contain death domain (DD) or immunoglobulin (Ig) domain typically located N-terminally to the p20 subunit. In contrast, all metacaspases harbor both p20 and p10 subunits and meanwhile lack DD and Ig domains. According to their distinct structural features, metacaspases are subdivided into three categories, namely Type I, Type II and Type III. Type-I metacaspases carry an ~90 AA-long sequence, termed prodomain, located N-terminally to the protease domain. The name of the prodomain presumably stems from the nomenclature for metazoan initiator caspases. However, the prodomain sequence of MCs not only differs from that of the initiator caspases but also varies markedly among different type-I metacaspase individuals (Klemencic and Funk, 2019). The function of the prodomain remains largely obscure so far. Type-II metacaspases are characterized by the absence of a prodomain, and a rather long linker region (~150 amino acid) that separates the p20 and p10 subunits. Type-III metacaspases feature an unusual organization of the protease 22 domain, with the p10 subunit preceding the p20. All green plant species examined so far appear to have both type-I and type-II MC genes in variable copy numbers, with three type-I MC-encoding genes (AtMC1-AtMC3) and six type-II (AtMC4-AtMC9) in A. thaliana (Fortin and Lam, 2018; Vercammen et al., 2004). The catalytic p20-like domain is colored in dark blue and the p10 domain in green. Additional domains are colored in light red. A dashed border indicates the possible presence of domains. The figure is not drawn to scale. Ig: immunoglobulin-like domain, DD death domain, N: N-terminal proline-rich repeat. This graph is adapted from Klemencic and Funk, 2018a. 1.5.4 Substrate specificity and calcium dependency of metacaspases To date, the crystal structure of two metacaspases, Saccharomyces cerevisiae Yca1 and Trypanosoma brucei TbMC2, has been resolved (McLuskey et al., 2012; Wong et al., 2012). Despite the overall structural similarity, metacaspases and caspases have some radical differences in their biochemical properties, primarily regarding the substrate specificity and Ca2+ dependency. Figure 1.3 Comparison of the structural organization of different groups of C14 family protease 23 Metacaspases have utterly distinct substrate specificity in comparison to caspases. Conventional caspase inhibitors are unable to block the activity of metacaspases and the tested metacaspases failed to cleave caspase-specific synthetic substrates in vitro (Vercammen et al., 2004). Accumulating biochemical evidence demonstrates that unlike caspases, which recognize the aforementioned tetrapeptide motif and perform cleavage C-terminally to an Asp, all three types of metacaspases show strict preference for peptide substrates with Arginine (R) or lysine (K) at the P1 position (Klemencic and Funk, 2018b; McLuskey et al., 2012; Vercammen et al., 2004). The explanation for such a striking distinction lies in the composition of their respective substrate-binding pocket. While caspases typically possess a basic specificity pocket, known MCs\u00E2\u0080\u0099 crystal structures display a substrate-binding pocket primarily composed of acidic residuals, explaining their R/K-P1 preference (McLuskey et al., 2012; Wong et al., 2012). Besides their distinct cleavage specificity, another stark distinction between metacaspase and caspase is the calcium dependency. In contrast to typical Ca2+-independent catalytic activity of caspases, the vast majority of metacaspases display strong reliance on the presence of calcium ion at the low millimolar range for full enzyme activity, despite a few exceptions like Arabidopsis MC9 (Klemencic and Funk, 2018b; Machado et al., 2013; Watanabe and Lam, 2011b). For example, in an in vitro protease assay, Arabidopsis type-II metacaspase MC4 cleaved the artificial metacaspase substrate Boc-GRR-MCA in a strictly Ca2+-dependent manner, as the addition of Ca2+ chelators abrogates its activity (Watanabe and Lam, 2011b). Site-directed mutagenesis disrupting the Ca2+ binding sites of a type-I metacaspase TbMC2 abolished its protease activity (McLuskey et al., 2012). Likewise, as a type-III metacaspase representative, Guillardia theta GtMC2 also undergoes Ca2+-dependent auto-processing and enzyme maturation (Klemencic and Funk, 2018b). 24 1.5.5 Controversy regarding the functional relevance of metacaspase prodomain As mentioned above, possession of an N-terminal prodomain (PD) is the primary defining trait of type-I metacaspases. This N-terminal prodomain is roughly 90 amino-acids long and usually rich in proline. Many prodomain sequences contain a zinc finger motif homologous to those found in LSD1, a well-known negative regulator of cell death (Lam and Zhang, 2012). The functional relevance of having a prodomain alongside an intact protease domain remains controversial. The overall prodomain sequence is highly diverse and there seems not to be a unifying functional explanation. One of the prevailing hypotheses arose from the examination of the TbMC2 crystal structure. The prodomain of TbMC2 circles across the Cys-His catalytic dyad and appears to prevent substrate entry to the catalytic site (McLuskey et al., 2012). Combined with the fact that the prodomain is often removed during the auto-processing step, a rational hypothesis takes shape that the prodomain serves as an autoinhibitory module to repress protease activity. However, using a site-directed mutagenesis approach, Gilio et al. (2017) found that a prodomain un-processable TbMC2 mutant retains its normal proteolytic activity against synthetic substrates, challenging the view that prodomain removal is a prerequisite for enzyme activation. In stark contrast to TbMC2, the Yca1 prodomain appears to have no detectable binding with its core protease structure (Wong et al., 2012). Consistently, the Yca1 prodomain-deletion mutant exhibits a similar level of in vitro peptidase activity compared to the wild-type form, indicating the Yca1 prodomain likely does not serve as an autoinhibitory domain (Wong et al., 2012). Similarly, the Arabidopsis MC1 is able to bind and cleave its substrate AtSerpin1 irrespective of the presence of its prodomain (Asqui et al., 2018). Alternatively, accumulating evidence suggests that prodomains may mediate the physical interaction of metacaspase with other signaling components. The Zinc finger motif has been shown 25 to mediate protein-protein interaction in many cases. For example, LSD1 interacts with MC1 through its zinc-finger domain in Y2H (Coll et al., 2010). Similarly, an intact zinc finger domain of LSD1 is also required for its interaction with bZIP10 in yeast (Kaminaka et al., 2006). Previous research also suggests that MC1 appeared to interact with itself and, in weaker strength, its prodomain in Y2H (Coll et al., 2010). The proline-rich repeats are also suggested to serve as potential platforms for protein-protein interaction (Opitz et al., 2015; Williamson, 1994). Hence, the prodomain of type-I metacaspase may enable novel potential protein-protein interaction capacity. 1.5.6 Regulatory roles of metacaspases in cell death and beyond Compared to animals, the molecular mechanisms governing the onset of plant programmed cell death (PCD) remain far more obscure. In recent decades, metacaspases, despite exhibiting the aforementioned biochemical differences from caspases, have stood out as critical players in controlling cell death triggered by diverse developmental and physiological cues (Lam and Zhang, 2012; Sueldo and van der Hoorn, 2017). However, in contrast to the definite role of caspases as apoptosis executors, plant metacaspases were shown to regulate cell death in both directions. For example, during plant embryogenesis, mcII-Pa, a type-II metacaspase from Picea abies facilitates the death of the cells destined for elimination in a catalytic activity-dependent manner (Bozhkov et al., 2005). Genetic interference of mcII-Pa function results in repressed PCD hallmarks in the embryos (Bozhkov et al., 2005). On the other hand, Arabidopsis type-II metacaspase MC9 is involved in the negative regulation of autophagy during vascular xylem differentiation (Escamez et al., 2016). Reduced expression of AtMC9 coincides with the accelerated death of tracheary 26 element cells and expansion of the autophagy zone, suggesting the native function of MC9 is to confine PCD to the xylem progenitor cells (Escamez et al., 2016). In addition to the development-related cell death, metacaspases are also implicated in death control under many different stress conditions. For instance, the yeast metacaspase Yca1 promotes the PCD triggered by acetic acid, hydrogen peroxide and hyperosmotic stress (Khan et al., 2005; Mazzoni and Falcone, 2008). Notable plant metacaspases examples are MC4 and MC1 from Arabidopsis. Characterization of mc4 knockout mutants reveals that MC4 is required for the onset of PCD induced by several abiotic and biotic stressors (Watanabe and Lam, 2011a). In a catalytic activity-dependent fashion, a type-I metacaspase MC1 positively regulates hypersensitive cell death during incompatible host-pathogen interactions (Coll et al., 2010). The mc1 null mutant is compromised in AvrRPM1-induced hypersensitive response and overexpression of MC1 results in ectopic cell death (Coll et al., 2010). Surprisingly, Arabidopsis MC2, a close homolog of MC1, negatively impacts the MC1-promoted cell death in a yet to be clarified mechanism (Coll et al., 2010). Oftentimes, a given metacaspase has multiple substrates; some substrates might be functionally unrelated to cell death regulation. By determining the proteolysis of these non-death-related substrates, metacaspases can also serve as critical regulators in pathways uncoupled from cell death control. One of the best-characterized examples is Arabidopsis MC9. Besides the aforementioned role in xylem differentiation, MC9 was shown to promote gluconeogenesis by activating a central enzyme of this pathway, phosphoenolpyruvate carboxykinase 1 (PEPCK1), via cleavage-dependent activation (Tsiatsiani et al., 2013). Dozens of potential in vivo substrates of MC9 were proposed in this proteome-wide degradome study, implicating broad functions of MC9 in cell physiology (Tsiatsiani et al., 2013). Another well-characterized multifaceted metacaspase 27 is the yeast Yca1. Besides its cell death role, Yca1 promotes cellular fitness via maintaining the normal progression of the cell cycle and preventing aberrant protein aggregation (Lee et al., 2010; Lee et al., 2008; Shrestha et al., 2019). Loss of Yca1 results in catastrophic outcomes such as disorganized cell division and the retention of insoluble protein materials due to excessive aggregation (Lee et al., 2010; Lee et al., 2008). Interestingly, Arabidopsis MC1 appeared to fulfill a similar role in protein aggregate management, underpinning a possible evolutionarily conserved function of metacaspases across kingdoms (Coll et al., 2014). Recent advances in the metacaspase field have uncovered their novel functions in regulating defense responses. For instance, in response to mechanical damage and herbivory, Arabidopsis MC4 initiates the proteolytic processing of endogenous precursors PROPEP1 in a Ca2+-dependent manner, thus releasing the mature form of elicitor-active pep1 (Hander et al., 2019; Shen et al., 2019). Mature pep1 is sensed by membrane receptors PEPR1/2 and elicits downstream immune responses (Yamaguchi et al., 2010). In an analogous scheme, MC9 is responsible for releasing the elicitor-active peptide from its precursor GRIM REAPER to induce ROS-dependent cell death (Wrzaczek et al., 2015). Interestingly, a type-II metacaspase from wheat (Triticum aestivum), TaMCA4, is rapidly induced when challenged by the stripe rust fungus P. striiformis f. sp. tritici, and contributes to the resistance against this leaf rust pathogen (Wang et al., 2012). Despite the progress achieved in recent years, research on metacaspase is still in its infancy. Novel regulatory roles of metacaspases await discovery and structure-function studies are needed to clarify the functional relevance of individual domains. Future crystal structure analysis of additional metacaspases will provide more mechanistic insights into their mode of action. 28 1.6 Thesis objective Finetuning the activity of pattern-recognition receptor signaling is vital for the well-being of plants, as the perturbation of major components of the PRR complex typically causes severe damage to plant growth and immunity. Plants have evolved sophisticated mechanisms to tightly control the heteromerization, phosphorylation and deactivation steps of the PRR complex. The objective of the present Ph.D. thesis is to identify and characterize novel mechanisms that finetune the activities of components of the PRR complex. The second chapter of my dissertation describes how the kinase activity and the immune function of the PAMP coreceptor BAK1 are intrinsically modulated by its carboxyl-terminal tail. The third chapter focuses on the functional studies of a type-I metacaspase in the regulation of PTI signaling mediated by BAK1, SOBIR1, and RLP-type PAMP receptors. 29 Chapter 2: The carboxyl-terminus tail (CT) of BAK1 is differentially required for its function in development and immunity 2.1 Summary In Arabidopsis, BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) is a multi-functional receptor-like kinase (RLK) that plays crucial roles in both plant developmental and immune signaling pathways. BAK1 and a large number of LRR-RLKs harbor a mysterious carboxyl-terminal tail (CT) beyond their kinase domain. In this study, we analyzed the biological significance of the BAK1 CT region using a bak1 mutant allele which contains a deletion of the CT. Our results demonstrate that the CT of BAK1 is required for its function in PAMP-triggered immunity (PTI), but is dispensable for cell death control and brassinosteroid-induced responses. Mechanistically, loss of CT drastically diminishes the autophosphorylation level of BAK1, suggesting the CT is an intrinsic regulatory module required for BAK1 kinase activity. This project reveals the differential requirement of BAK1\u00E2\u0080\u0099s C-terminal tail in development and immunity. 2.2 Introduction Multicellular plants need to optimize both cell-to-cell communication and cell-to-environment communication to thrive in a highly fluctuating natural environment. The capacity to sense extracellular stimuli is vital for plant growth, adaptation, and response to environmental and biological stresses. Hence, plants have evolved a huge number of surface-localized receptors such as receptor-like kinases (RLKs) to perceive diverse types of extracellular signal molecules (Ma et al., 2016a; Macho and Zipfel, 2014). Plant RLKs have a structural configuration similar to metazoan receptor tyrosine kinases, typically consisting of a unique extracellular domain for ligand 30 binding, a transmembrane domain, and a cytoplasmic kinase domain (Shiu and Bleecker, 2001). The LRR-RLK family, whose members carry extracellular leucine-rich repeat (LRR) domains, has more than 200 members, making LRR-RLKs the most predominant type of RLK (Shiu and Bleecker, 2001). Plant RLKs regulate diverse developmental, physiological, and immune signaling pathways. One famous example is the Arabidopsis BAK1, also known as SERK3 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 3). In brassinosteroid (BR) signaling, BAK1 works together with BRI1 (BRASSINOSTEROID INSENSITIVE 1) to perceive BR (Li et al., 2002a; Nam and Li, 2002). In pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI), BAK1 serves as a co-receptor for multiple RLK-type and receptor-like protein (RLP)-type pattern recognition receptors (PRRs) (He et al., 2018). In addition, BAK1 functions redundantly with its close homolog BKK1 (BAK1-LIKE 1) in cell death control (He et al., 2007). Structurally speaking, BAK1 contains an extracellular LRR domain, a transmembrane helix and a cytoplasmic kinase domain followed by a carboxyl-terminal tail (CT). The kinase activity of BAK1 has been demonstrated to be required for its functions in BR signaling and plant immunity (Nam and Li, 2002; Schulze et al., 2010; Schwessinger et al., 2011). Interestingly, a missense mutation in bak1-5 that results in a single amino acid change in the kinase domain and partial loss of the kinase activity of BAK1 has a more profound effect on blocking PTI signaling than the bak1-4 knockout mutant, but has no effects on its functions in BR signaling and cell death control, suggesting that bak1-5 specifically interferes with PTI signaling (Roux et al., 2011a; Schwessinger et al., 2011). Regarding the CT of BAK1, very little is known about its role in plant development and immunity, even though the presence of CTs in BAK1 and other SERKs and a 31 high degree of sequence conservation among these CTs have been documented previously (Aan den Toorn et al., 2015; Schwessinger and Rathjen, 2015). Here, we uncovered the functional significance of the BAK1 CT in different BAK1-mediated signaling pathways, by using a bak1 mutant allele that deletes the CT region. Our data demonstrate that the BAK1 CT promotes its kinase activity, and is required for BAK1\u00E2\u0080\u0099s function in PTI signaling but is dispensable in cell death control and BR-mediated developmental process. Furthermore, a survey of all putative LRR-RLKs in A. thaliana reveals that CT extension is broadly present throughout the LRR-RLK family, suggesting that CT extension may also regulate the function of many other LRR-RLKs. 2.3 Materials and methods 2.3.1 Growth conditions and mutant generation Arabidopsis plants were grown on soil at 22 \u00E2\u0084\u0083 under long-day conditions (16-hr light/8-hr dark) unless otherwise stated. When growing Arabidopsis under sterile conditions, stratified seeds were plated on half-strength Murashige and Skoog medium (1/2 MS), after which plates were incubated in a growth chamber under long-day conditions (16-hr light at 22 \u00E2\u0084\u0083/8-hr dark at 19 \u00E2\u0084\u0083). The sobir7-1 bir1-1 pad4-1 triple mutant was previously described (Liu et al., 2016). To generate the sobir7-1 single mutant, sobir7-1 bir1-1 pad4-1 was crossed with Col-0 wild type. To generate the sobir7-1 bkk1-1 double mutant, sobir7-1 bir1-1 pad4-1 was crossed with bak1-4+/- bkk1-1. The desired mutants were subsequently isolated from the respective F2 population through PCR-based genotyping with specific primers summarized in Table 2.1. 32 2.3.2 Plasmid construction The full-length BAK1 DNA sequence was amplified with Phusion DNA Polymerase (Fisher Scientific International, Inc.) using specific primers in Table 2.1. The resulting PCR fragments were digested and then ligated into different expression vectors according to experimental design. Specifically, for transient expression in Nicotiana benthamiana (hereafter N. benthamiana), the full-length BAK1 DNA sequence was ligated into a modified pCambia1300 vector containing a 35S promoter and C-terminal hemagglutinin (HA) tag. For the bimolecular fluorescence complementation assay, the full-length BAK1 cDNA was incorporated into a pUC19-YCE vector. For heterogeneous expression of the BAK1 kinase domain (KD) in E. coli, the BAK1 kinase domain cDNA sequence was ligated into a pET24c vector for the production of 6\u00C3\u0097His-tagged BAK1 KD protein in the bacteria. Subsequently, the same set of constructs was generated for the mutant BAK1 carrying the sobir7-1 mutation with the corresponding wild-type BAK1 plasmids as templates, using specific primers listed in Table 2.1. 2.3.3 Total protein extraction and western blot Approximately 60 mg A. thaliana or N. benthamiana tissue was ground thoroughly and homogenized in two volumes (2 \u00C2\u00B5l per mg) 2\u00C3\u0097SDS extraction buffer (100 mM pH 6.8 Tris-HCl, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM DTT) to obtain total plant protein lysate. After boiling for 5 min twice, the extract was spun down at 15,000 g for 5 min to pellet the cell debris. The supernatant was then collected and loaded for western blot analysis. 33 2.3.4 Measurement of PAMP-triggered reactive oxygen species (ROS) Measurement of PAMP-triggered ROS production was performed as described previously (Liu et al., 2013a). Essentially, leaf strips of 4-week-old short-day grown Arabidopsis plants were treated with an elicitor solution consisting of 20\u00E2\u0080\u0089\u00CE\u00BCM luminol and 10 mg/ml horseradish peroxidase, and 1 \u00C2\u00B5M elicitor peptide (flg22 or elf18). Chemiluminescence was then monitored by a microplate reader over a period of one hr and the peak value of each group was used for presentation (Tecan Group Ltd., Tecan M200). 2.3.5 PAMP-triggered activation of MAP kinases To measure flg22-triggered MAP kinase activation, two-week-old Col-0, bak1-4 and sobir7-1 seedlings grown on \u00C2\u00BD MS medium were thoroughly sprayed with 100 nM flg22 solution supplemented with 0.02% silwet. Seedling samples were pooled at both 0 min and 15 min post-treatment. Subsequently, phosphorylated MAPKs were detected by anti-phospho-p44/42-ERK antibody (Cell Signaling Technology, Inc., #9102). 2.3.6 RNA extraction and quantitative reverse transcription PCR (qPCR) For gene expression analysis, 12-day-old seedlings grown on \u00C2\u00BD MS plates were pooled for RNA extraction with an EZ-10 Spin Column Plant RNA Mini-Preps Kit (Bio Basic, Canada) following manufacturers\u00E2\u0080\u0099 instructions. The first strand of cDNA was synthesized by reverse transcriptase with the EasyScript\u00E2\u0084\u00A2 Reverse Transcriptase (ABM, Canada), after which real-time PCR was performed with the SYBR Premix Ex TaqII kit (Takara, Japan) to quantify the transcript level of target genes. 34 2.3.7 Measurement of Arabidopsis hypocotyl growth To measure hypocotyl elongation under Epibrassinolide (Sigma-Aldrich, E1641) treatment, Arabidopsis seeds were plated on \u00C2\u00BD MS medium solidified with 12g/L agar and supplemented with 100 nM Epibrassinolide or the mock solution. Plates were relocated into a light chamber for two days to allow for efficient germination. The plates were subsequently kept vertically in the dark for another six days before measuring hypocotyl length. At least 30 seedlings from each experimental group were randomly chosen for hypocotyl measurement. 2.3.8 Bimolecular fluorescence complementation (BiFC) assay All plasmids used in the BiFC assay were purified through the PureYield\u00E2\u0084\u00A2 Plasmid Midiprep System (Promega Corporation). Preparation of Arabidopsis mesophyll cell protoplasts has previously been described (Wu et al., 2009). Protoplasts were transfected via a polyethylene glycol (PEG)-mediated transformation approach and then incubated under weak light for approximately 16 hrs. Following incubation, protoplasts were harvested by low-speed centrifugation and then examined with a Nikon ECLIPSE 80i confocal microscope to detect fluorescence. 2.3.9 Trypan blue staining Trypan Blue staining was carried out following previously described protocols (ThordalChristensen et al., 1997). Essentially, seedlings were immersed into lactophenol Trypan Blue work solution (10 mg Trypan Blue, 10 g phenol, 10 ml lactic acid, 10 ml glycerol and 10 ml water, diluted 1:1 in ethanol to make work solution). After boiling for 3 minutes, the staining solution was removed and samples were then de-stained using 2 ml chloralhydrate solution (2.5 35 g/ml chloralhydrate) with gentle shaking for 2 hrs. Samples were further de-stained overnight with a new chloralhydrate solution before examination with a light microscope (Olympus Stereo Microscope SZX 10). 2.3.10 Purification of His-tagged BAK1 kinase domain from E. coli Constructs expressing the BAK1 kinase domain (BAK1-KD) and SOBIR7-1 kinase domain (SOBIR7-1-KD) were transformed into E. coli strain BL21 (New England BioLabs), respectively. The transformed E. coli strains were grown in 500 ml liquid Lysogeny broth (LB) to reach an approximate density of OD600 = 0.4. Afterward, expression of the transgene was induced by 0.4 mM IPTG at 17 \u00E2\u0084\u0083 for 16 hrs. Following induction, bacteria were harvested by centrifugation and then re-suspended in lysis buffer consisting of 25mM Tris-HCl (pH 8.0) and 150 mM NaCl and 1 mM PMSF. Bacteria cells were then lysed by sonication, after which the resulting cell lysate was filtered through Ni-NTA affinity resin (QIAGEN N.V.). The Ni-NTA matrices were then thoroughly rinsed with wash buffer (25 mM pH 8.0 Tris-HCl, 150 mM NaCl, 15 mM imidazole) before His-tagged protein was eluted with 2 ml elution buffer (25 mM pH 8.0 Tris-HCl, 150 mM NaCl, 250 mM imidazole). Eluted protein was subjected to dialysis prior to storage at -80 \u00E2\u0084\u0083. In the subsequent immunoblotting assay to determine the autophosphorylation status of BAK1-KD and SOBIR7-1-KD, approximately 1 \u00C2\u00B5g protein was loaded during gel electrophoresis. Anti-phospho-Ser/Thr antibody (BD Transduction Laboratories\u00E2\u0084\u00A2) was used to probe the western blot membrane to determine the autophosphorylation level of BAK1-KD and SOBIR7-1-KD. 36 2.3.11 Statistical analysis Statistical comparison of the mean value among different samples was conducted with Student\u00E2\u0080\u0099s t-test or one-way ANOVA test in RStudio software (Version 1.1.456 \u00E2\u0080\u0093 \u00C2\u00A9 2009-2018 RStudio, Inc.). The p-values of statistical comparison are provided in the legends of their respective figures. Stars (*) are used to show statistically significant differences between two distinct samples. For comparisons involving three or more samples, different letters (a, b, c, etc.) are used to label the samples with statistically significant differences; whereas the same letter was used to mark the samples without a significant difference. Table 2.1 Primers used in Chapter 2 Primer Name Sequence (5\u00E2\u0080\u0099 to 3\u00E2\u0080\u0099) Function BAK1-pG229HAN-Kpn1-F CGGGGTACCATGGAACGAAGATTAATGATCCCTTG Cloning pCambia1300-35S-BAK1-3\u00C3\u0097HA and pCambia1300-35S-SOBIR7-1-3\u00C3\u0097HA, pairing with BAK1-StuI-R and tBAK1-StuI-R, respectively BAK1-StuI-R CGCGGATCCTTATCTTGGACCCGAGGGGTATTC Cloning pCambia1300-35S-BAK1-3\u00C3\u0097HA tBAK1-StuI-R GAAGGCCTGCCAGACACGGCTGGATG Cloning pCambia1300-35S-SOBIR7-1-3\u00C3\u0097HA BAK1-BiFC-BamHI-XhoI-F CGGGGATCCCTCGAGATGGAACGAAGATTAATGATCC Cloning BAK1-pUC19-YCE and SOBIR7-1-pUC19-YCE, pairing with BAK1-BiFC-XhoI-R and Bak1W644KpniR, respectively BAK1-BiFC-XhoI-R CGGCGGCTCGAGTCTTGGACCCGAGGGGTATTCG Cloning BAK1-pUC19-YCE 37 Bak1W644KpniR CGGGGTACCGCCAGACACGGCTGGATG Cloning SOBIR7-1-pUC19-YCE Bak1-NdeI-F GGAATTCCATATGGGACAACTGAAGAGGTTTTCATTG Cloning BAK1-KD-pET24c and SOBIR7-1-KD-pET24c, pairing with BAK1-BiFC-XhoI-R and tBAK1-XhoI-R, respectively BAK1-BiFC-XhoI-R CGGCGGCTCGAGTCTTGGACCCGAGGGGTATTCG Cloning for BAK1-KD-pET24c tBAK1-XhoI-R CGGCGGCTCGAGGCCAGACACGGCTGGATG Cloning for SOBIR7-1-KD-pET24c WtSobir7_1F2 TCCAGCCGTGTCTGGCAGG Genotyping for sobir7-1 SNP, pairing with BAK1-s1_F BAK1_s1_F CATGAATCTTCTAGGCTACTATG Genotyping for sobir7-1 SNP, pairing with WtSobir7_F2 Bak1-4-F GGCCACTAAAGTACCATCAG Genotyping for bak1-4 T-DNA homozygote, pairing with Bak1-4-R Bak1-4-R CCTCTCACCGGAGATATTCCT Genotyping for bak1-4 T-DNA homozygote, pairing with Bak1-4-F P745 AACGTCCGCAATGTGTTATTAAGTTGTC Genotyping for bir1-1 T-DNA presence, paring with BIR1-179-F BIR1-179-F AGAACGCAGTTGCATGCTAC Genotyping for bir1-1 T-DNA presence, pairing with P745 BKK1-1-NF CCAGCCATTGCGTTTGCTTG Genotyping for bkk1-1 T-DNA homozygote, pairing with BKK1-1-NR BKK1-1-NR GCGTACAGCAGTTGTCACA Genotyping for bkk1-1 T-DNA homozygote, pairing with BKK1-1-NF pad4-1-MT-F GCATAAGACTAGCTAAGTTTCA Genotyping for pad4-1 presence, paring with pad4-1-R 38 pad4-1-R AAGTCTCCATTGCGTCACTC Genotyping for pad4-1 presence, paring with pad4-1-MT-F Lba1 TGGTTCACGTAGTGGGCCATCG T-DNA Genotyping 2.4 Results 2.4.1 The sobir7-1 mutation causes deletion of the carboxyl-terminal tail of BAK1 From a previous bir1-1 pad4-1 suppressor screen (SOBIR: Suppressor of bir1-1), we identified sobir7-1, a bak1 allele that harbors a nonsense mutation within the BAK1 CT leading to almost complete deletion of the CT (Figure 1.1A), suggesting that the CT of BAK1 is required for its function in promoting cell death in the bir1-1 mutant (Liu et al., 2016). Interestingly, sobir7-1 suppresses bir1-1 pad4-1 more dramatically than the bak1-4 null allele (Liu et al., 2016), suggesting that the mutant protein may interfere with the function of its close homologs such as BKK1. Analysis of F1 plants from a cross between sobir7-1 bir1-1 pad4-1 and bir1-1 pad4-1 showed that the F1 plants exhibited intermediate dwarf morphology compared to the parents (Figure 2.1A), suggesting that sobir7-1 is a semi-dominant mutation with a dominant-negative effect. 39 (A) Schematic representation of BAK1 protein structure. The sobir7-1 mutation indicated by the arrowhead converts W597 to stop codon. LRR, leucine-rich repeats; TM, transmembrane helix; KD, kinase domain. CT, carboxyl-terminal tail. (B) Morphology of an F1 plant generated by crossing bir1-1 pad4-1 (b. p.) with sobir7-1 bir1-1 pad4-1 (sobir7-1 b. p.). (C) Crossing schemes to obtain sobir7-1 single and sobir7-1 bkk1-1 double mutants. 2.4.2 BAK1 CT is not required for its function in cell death control The identification of the sobir7-1 allele offered us a unique tool to analyze the function of BAK1 CT in developmental and immune signaling. We, therefore, crossed sobir7-1 bir1-1 pad4-1 with Col-0 and bak1-4+/- bkk1-1 to obtain the sobir7-1 single and sobir7-1 bkk1-1 double mutants (Figure 2.1C). To determine the function of the BAK1 CT in cell death control, we compared the morphology of sobir7-1 bkk1-1 with bak1-4 bkk1-1. Unlike bak1-4 bkk1-1, sobir7-1 bkk1-1 Figure 2.1 The sobir7-1 mutation leads to deletion of Carboxyl-terminal tail (CT) of BAK1. 40 displayed wild-type-like morphology (Figure 2.2A). Trypan blue staining showed that the spontaneous cell death observed in bak1-4 bkk1-1 is absent in sobir7-1 bkk1-1 (Figure 2.2B). Consistently, the expression of the defense marker gene PR2 in sobir7-1 bkk1-1 is not constitutively activated (Figure 2.2C). Collectively, these data suggest that the CT is not required for BAK1\u00E2\u0080\u0099s function in suppressing spontaneous cell death. (A) Morphology of two-week-old plate-grown seedlings of wild type (WT), bak1-4 bkk1-1 and sobir7-1 bkk1-1. Scale bar = 1 cm. (B) Trypan blue staining of two-week-old WT, bak1-4 bkk1-1, and sobir7-1 bkk1-1 seedlings. Scale bar = 0.5 mm. (C) PR2 expression in WT, bak1-4, sobir7-1, bkk1-1, bak1-4 bkk1-1 and sobir7-1 bkk1-1. Error bars represent the standard deviation of three technical replicates. Data are representative of three independent experiments. Figure 2.2 BAK1 CT is dispensable for its function in suppressing spontaneous cell death. 41 2.4.3 The sobir7-1 and sobir7-1 bkk1-1 mutants are compromised in the PAMP-triggered immunity We next analyzed PTI responses in sobir7-1 and sobir7-1 bkk1-1. As shown in Figure 2.3A and B, respectively, flg22- and elf18-induced production of reactive oxygen species (ROS) was completely blocked in sobir7-1 and sobir7-1 bkk1-1. In sobir7-1, flg22-induced MAP kinase activation was also clearly reduced (Figure 2.3C). We also examined the transcription upregulation of a PTI marker gene FRK1 by nlp20, a PAMP which also triggers BAK1-dependent defense signaling. We found the nlp20-induced FRK1 expression was abrogated in sobir7-1 and sobir7-1 bkk1-1 mutants (Figure 2.3D). Moreover, the growth of Pseudomonas syringe pv. tomato (P.s.t.) DC3000 hrcC, a strain deficient in the type-III secretion system, was significantly higher in sobir7-1 and increased more dramatically in the sobir7-1 bkk1-1 double mutant (Figure 2.3E). Taken together, these data indicate that the CT is required for the function of BAK1 in PTI. 42 43 (A) Measurement of flg22-induced reactive oxygen species production in WT, bak1-4, sobir7-1 and sobir7-1 bkk1-1 mutants. Different letters indicate statistical difference (p < 0.05, one-way ANOVA). Error bars represent the standard deviation of at least eight biological replicates. This assay was repeated three times with similar results. (B) Measurement of elf18-induced reactive oxygen species production in WT, bak1-4, sobir7-1 and sobir7-1 bkk1-1 mutants. Error bars represent the standard deviation of six biological replicates. This assay was performed twice with similar results. (C) Immunoblotting showing flg22-induced mitogen-activated protein kinase (MAPK) activation in the sobir7-1 mutant. (D) Expression of FRK1 in WT, bak1-4, sobir7-1, sobir7-1 bkk1-1 before and 4 hrs after treatment of 100 nM nlp20. Statistical comparison was performed among samples collected at the same timepoint. Different letters mark samples with significant difference (p < 0.01, one-way ANOVA) while samples without statistical difference are labeled by \u00E2\u0080\u0098n.d.\u00E2\u0080\u0099 (p < 0.05). (E) Growth of Pseudomonas syringe pv. tomato (P.s.t.) DC3000 hrcC on WT, bak1-4, sobir7-1 and sobir7-1 bkk1-1 mutants at 3 days post-inoculation (Day 3). Different letters indicate statistical differences determined by one-way ANOVA (p < 0.05, N \u00E2\u0089\u00A5 6). 2.4.4 BAK1 CT is not essential for brassinosteroid (BR) signaling To determine whether sobir7-1 affects the brassinosteroid (BR) signaling, we examined the morphology of sobir7-1 adult plants grown under short-day conditions. Unlike the bak1-4 control, which has a smaller rosette and shortened petiole, the sobir7-1 mutant did not exhibit any clear growth defect relative to wild type (Figure 2.4A). We then investigated whether the growth of etiolated sobir7-1 seedlings is inhibited by brassinolide (BL, a bioactive BR product) treatment. Similar to wild type, hypocotyl elongation in sobir7-1 was inhibited by BL (Figure 2.4B), Figure 2.3 The sobir7-1 single and sobir7-1 bkk1-1 double mutants are defective in pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI) responses. 44 suggesting the CT-deletion BAK1 variant is likely signaling-competent in the BR pathway. Collectively, our data indicate that sobir7-1 does not block BR responses and the BAK1 CT is not essential for BR signaling. (A) Morphology of four-week-old sobir7-1 mutant grown under short-day conditions. Scale bar = 1 cm. (B) Brassinolide (BL)-induced hypocotyl growth inhibition of WT, bak1-4 and sobir7-1 seedlings. The asterisks indicate statistical difference (p < 0.01, T-Test, N \u00E2\u0089\u00A5 30). Samples without statistical differences are marked by \u00E2\u0080\u0098n.d.\u00E2\u0080\u0099. This assay was repeated twice with similar results. Figure 2.4 BAK1 CT is not essential for brassinosteroid (BR) signaling. 45 2.4.5 Loss of CT does not affect the protein stability of BAK1 To understand the molecular mechanism behind the function of BAK1 CT, we transiently expressed hemagglutinin (HA)-tagged full-length BAK1 and the truncated SOBIR7-1 mutant protein in N. benthamiana. As shown in Figure 2.5, SOBIR7-1-HA protein accumulated to a similar level to BAK1-HA, suggesting that loss of the CT does not affect the stability of BAK1 protein. Immunoblotting with anti-HA antibody to examine the protein accumulation of hemagglutinin (HA)-tagged BAK1 and SOBIR7-1 transiently expressed in N. benthamiana. The expression of both BAK1-HA and SOBIR7-1-HA was controlled by 35S promoters. 2.4.6 Deletion of the CT of BAK1 does not compromise its capacity to interact with FLS2 and BIK1 We then tested whether the interaction between BAK1 and other signaling components, mainly FLS2 (FLAGELLIN-SENSITIVE 2) and BIK1 (BOTRYTIS-INDUCED KINASE1), is affected by the lack of the CT. In split luciferase assay, the tail-deletion mutant SOBIR7-1 still associated with FLS2 as wild-type BAK1 does, indicating the PTI defects of the sobir7-1 mutant were not due to a loss of FLS2-BAK1 dimerization (Figure 2.6A, B). Figure 2.5 Loss of CT does not affect the protein stability of BAK1. 46 A representative N. benthamiana leaf showing the interaction between FLS2 and BAK1 or SOBIR7-1 in the split luciferase assay. The graph on the right indicated different combinations of testing. The NPR3-Nluc + TGA2-Cluc group was used as a positive control. The assay was performed twice with similar results. Consistent with a previous report that BAK1 interacts with BIK1 (Lu et al., 2010a), we detected the interaction between BIK1 and BAK1 in both bimolecular fluorescence complementation (BiFC) and split luciferase assay (Figure 2.7A, B). The sobir7-1 mutation does not affect this interaction, suggesting that the PTI defect in sobir7-1 is not due to compromised interaction between BAK1 and BIK1. Figure 2.6 Loss of CT of BAK1 does not compromise its ability to interact with FLS2 in split luciferase assay. 47 (A) Bimolecular fluorescence complementation assay showing the interaction between BAK1 or SOBIR7-1with BIK1. Scale bars = 10 \u00C2\u00B5m. Figure 2.7 The SOBIR7-1 mutant protein retains the ability to associate with BIK1. 48 (B) Split luciferase assay showing the quantitative results of the interaction between BAK1 or SOBIR7-1 with BIK1. Different letters indicate statistically significant differences (p < 0.05, N \u00E2\u0089\u00A5 8). 2.4.7 The CT region is required for the kinase activity of BAK1 Next, we tested whether the BAK1 kinase activity is influenced by the sobir7-1 mutation. We purified the C-terminal His-tagged BAK1 cytoplasmic domain (BAK1-KD) and SOBIR7-1 cytoplasmic domain (SOBIR7-1-KD) from E. coli. The auto-phosphorylation strength of the recombinant proteins was then analyzed by immunoblotting with an anti-phospho-Ser/Thr antibody. As shown in Figure 2.8A, auto-phosphorylation intensity of SOBIR7-1-KD was dramatically reduced compared to wild-type BAK1-KD, suggesting that loss of CT leads to reduced BAK1 autophosphorylation. When we aligned the CTs of BAK1 and other LRR-RLKs in subfamily II, we found a conserved \u00E2\u0080\u0098SGPR\u00E2\u0080\u0099 motif at the carboxyl end (Figure 2.8B). This motif is also highly conserved in the BAK1 homologs in tomato (S. lycopersicum), tobacco (N. benthamiana.), and rice (O. sativa), but it is not conserved outside subfamily II. To test whether it is required for the kinase activity of BAK1, we expressed the BAK1-KD lacking the last three amino acids (BAK1-KD-\u00CE\u0094GPR) in E. coli. Western blot analysis using the anti-phospho-Ser/Thr antibody showed that there is a dramatic reduction of auto-phosphorylation in BAK1-KD-\u00CE\u0094GPR compared to BAK1-KD (Figure 2.8C), suggesting that the \u00E2\u0080\u0098GPR\u00E2\u0080\u0099 motif in the BAK1 CT is important for the kinase activity. 49 (A) Immunoblotting analysis of the autophosphorylation activity of the cytoplasmic domain of BAK1 (BAK1-KD) and the CT-deletion mutant SOBIR7-1 (SOBIR7-1-KD). Approximately 1 \u00C2\u00B5g of purified protein corresponding to the intracellular domain of wild-type BAK1 or SOBIR7-1 were subjected to western blot analysis with an anti-phospho-Ser/Thr (\u00CE\u00B1-pS/pT) antibody. The bottom panel indicates the relative band intensity. WB, western blot; CBS, Coomassie Blue staining. (B) Multiple alignment of the CT sequence from BAK1 and its homologs. Bracket represents the conserved \u00E2\u0080\u0098GPR\u00E2\u0080\u0099 motif. Asterisks indicate the previously identified two serine autophosphorylation sites of the BAK1 intracellular domain. The left column lists the gene Figure 2.8 The CT of BAK1 promotes its autophosphorylation activity. 50 name of BAK1 homologs in Arabidopsis and other plant species. Specifically, \u00E2\u0080\u0098At\u00E2\u0080\u0099 represents Arabidopsis thaliana; \u00E2\u0080\u0098AL\u00E2\u0080\u0099 is Arabidopsis lyrata; \u00E2\u0080\u0098Bradi\u00E2\u0080\u0099 is Brachypodium distachyon; \u00E2\u0080\u0098Os\u00E2\u0080\u0099 is Oryza sativa (Rice); \u00E2\u0080\u0098Soly\u00E2\u0080\u0099 is Solanum lycopersicum (Tomato); \u00E2\u0080\u0098Nb\u00E2\u0080\u0099 is Nicotiana benthamiana (Tobacco). (C) Immunoblotting analysis of the autophosphorylation of BAK1, SOBIR7-1, and the BAK1 mutant lacking the \u00E2\u0080\u0098GPR\u00E2\u0080\u0099 motif (BAK1-KD-\u00CE\u0094GPR) with an \u00CE\u00B1-pS/pT antibody. 2.4.8 The CT extension is broadly distributed throughout the LRR-RLK family The CT of another RLK, BRI1 (BRASSINOSTEROID INSENSITIVE 1), was previously shown to negatively regulate its kinase activity and function (Wang et al., 2005). To determine whether CTs are commonly present in LRR-RLKs, we examined 232 predicted LRR-RLKs in Arabidopsis for the presence of CTs. Interestingly, the CTs are widely spread among LRR-RLK families, with approximately 60% LRR-RLKs harboring a recognizable CT (Figure 2.9A), suggesting that the CT may play a general role in regulating the function of a wide array of LRR-RLKs. Notable CT-containing representatives are EFR, an important PAMP receptor; ERECTA, a receptor of endogenous EPF peptides involved in stomata patterning; TMK1, a key player in auxin signaling; and CIK3, a coreceptor involved in the shoot meristem maintenance pathway (Figure 2.8B). Intriguingly, a CT is present in all members of subfamily II, IV, VIII, and IX, but it is absent among a number of LRR-RLKs involved in plant immunity, such as FLS2, SOBIR1, and PEPR1/2. There is also no correlation between the length of the extracellular LRR domain and the presence of a CT. For example, despite carrying a short LRR like BAK1, SOBIR1 does not exhibit 51 a recognizable CT. Although both carry long LRR domains, EFR harbors a CT extension but FLS2 does not. 52 (A) The distribution of CT extensions among all the LRR-RLK subfamilies. (B) A shortlist of CT-containing LRR-RLKs that play important roles in plant growth and defense. 2.5 Discussion The multifaceted functions of BAK1 in plant development and immunity have been elucidated by genetic studies over the past decades (Chinchilla et al., 2007; He et al., 2007; Li et al., 2002a). Structurally speaking, BAK1 is composed of an LRR ectodomain, a transmembrane region, an intracellular kinase domain, and a C-terminal tail (CT) extension. The relationships between BAK1 function and its ectodomain as well as its kinase domain have been extensively explored by structure-function analysis (Nam and Li, 2002; Schulze et al., 2010; Sun et al., 2013a; Sun et al., 2013b). However, the functional significance of the CT region still remains mysterious, despite early documentation of its presence and its sequence conservation (Aan den Toorn et al., 2015; Schwessinger and Rathjen, 2015). Here, through phenotypic characterization of a bak1 mutant in which the CT is deleted by a premature stop codon, we clarified the functional requirement of the BAK1 CT in innate immunity, steroid hormone signaling, and cell death control. We found that the BAK1 CT is essential for PTI but does not play a significant role in cell death inhibition and BR-mediated plant growth, suggesting the differential requirement of the BAK1 CT in distinct biological pathways. Our mechanistic studies revealed that the CT of BAK1 is required for its full kinase activity, as evidenced by a drastically reduced autophosphorylation level in the CT-deletion mutant SOBIR7-1 (Figure 2.8B). Remarkably, there are indeed four verified phosphorylation sites inside Figure 2.9 The CT extensions are widely distributed throughout the leucine-rich repeat receptor-like kinase (LRR-RLK) family. 53 BAK1 CT: S602, T603, S604 and S612 (Karlova et al., 2009; Perraki et al., 2018). Thus, the reduced autophosphorylation may be directly caused by the loss of these phosphosites in the SOBIR7-1 mutant variant. In support of this idea, a recent publication reported that mutating these four Ser/Thr sites simultaneously into Ala strongly undermined the overall autophosphorylation level of BAK1, and, mimicking the sobir7-1 mutation, specifically impairs PTI responses but has no effect on BR signaling or cell death inhibition (Perraki et al., 2018). However, our data showed that removal of the conserved \u00E2\u0080\u0098GPR\u00E2\u0080\u0099 motif alone also has a dramatic impact on BAK1 autophosphorylation level, suggesting that in addition to carrying phosphosites, CT plays a role in establishing proper conformation of BAK1. One possibility is that the BAK1 CT serves as an intrinsic stimulator that helps to stabilize the kinase domain in an active conformation; thus, loss of CT will then render the kinase domain hypoactive. It would be very interesting to investigate whether, and if so, how the introduction of the BAK1-KD-\u00CE\u0094GPR variant into the bak1 null mutant affects distinct BAK1-regulated signaling pathways. Future crystal structure analysis of full-length BAK1 will also provide structural insights into the mode of action of the CT extension. Interestingly, the BAK1 CT functions in a manner opposite to the carboxyl terminus of some metazoan receptor tyrosine kinases and Arabidopsis BRI1, where the presence of the CT inhibits kinase activity (Schlessinger, 2000; Wang et al., 2005). It seems that the CT extension of different membrane receptors may have distinct functions. This is not surprising given that the BAK1 CT sequence is poorly conserved compared to BRI1 CT (data not shown). Similar to sobir7-1, the bak1-5 mutation located in the kinase domain also causes reduced kinase activity and a strong defect in PTI but does not affect the function of BAK1 in cell death control and BR signaling (Roux et al., 2011a; Schwessinger et al., 2011). In both cases, the differential impact of bak1 mutation on BR1-dependent and FLS2/EFR-dependent signaling 54 appears to be associated with reduced BAK1 kinase activity. The explanation might be a consequence of different degrees of reliance on BAK1 kinase activity by distinct receptors for signaling activation. In support of this notion, BRI1 was shown to be far more active in vitro than FLS2 and EFR (Schwessinger et al., 2011). When kinase activity of BAK1 is reduced, as seen in bak1-5, the BAK1-BRI1 trans-phosphorylation occurred more or less normally but the BAK1-EFR mutual phosphorylation was greatly compromised (Schwessinger et al., 2011). This could also be the case for the sobir7-1 allele. The residual kinase activity of SOBIR7-1 is likely sufficient for activation of the BRI1-BAK1 growth receptor complex but not enough for immune receptors such as FLS2/EFR. Previously, it was shown that adding C-terminal tags to BAK1 impairs its function in PTI signaling but not BR signaling (Ntoukakis et al., 2011). Such C-terminal tagging effects mimic the consequences of CT deletion, suggesting that C-terminal tags potentially interfere with the activity of the BAK1 CT, possibly through steric hindrance. Future crystal structure analysis of the BAK1 intracellular domain will help to clarify this hypothesis. When measuring PAMP-induced ROS production and FRK1 expression, sobir7-1 showed more pronounced defects than the null allele of BAK1, bak1-4 (Figure 2.3A, B, D), suggesting a dominant-negative effect of the sobir7-1 mutation. This dominant effect indicates that the SOBIR7-1 protein may interfere with the activity of BKK1 in planta, which has been shown to function redundantly with BAK1 in PTI (Roux et al., 2011a). Future studies are needed to determine the molecular mechanism behind this dominant-negative effect. Our survey of all putative LRR-RLKs in Arabidopsis reveals that a CT extension is broadly present throughout the LRR-RLK subfamilies, suggesting that CTs may also orchestrate signaling pathways mediated by other LRR-RLKs, possibly also via modulating their kinase activity. In 55 support of this idea, a recent study points out that CTs deletions of several Arabidopsis LRR-RLKs and Brassica oleracea BAK1 ortholog, could impact the kinase activity of their respective proteins in vitro (Oh et al., 2018). Furthermore, CT-containing LRR-RLKs include some key regulators controlling stomata patterning (e.g., ERECTA), growth phytohormone signaling (e.g., TMK1), floral organ abscission (e.g. HAESA) and stress responses (e.g. PRK5). Future investigation into the functions of these CT extensions will further deepen our understanding of the sophisticated regulatory mechanism that fine-tunes the cellular activity of these crucial membrane receptors. 56 Chapter 3: The prodomain of Arabidopsis Metacaspase 2 positively regulates immune signaling mediated by pattern-recognition receptors 3.1 Summary Metacaspases (MCs) are structural homologs of mammalian caspases found in plants, fungi, and protozoa. Type-I MCs carry an N-terminal prodomain, the function of which is unclear. MC2 is one of the three type-I MCs in Arabidopsis. Here, taking advantage of mc2-1, an autoimmune mutant of MC2, we uncovered a function of MC2 in promoting PAMP-triggered immunity (PTI) signaling. Pathogen infection strongly induces the expression of MC2 and elevated expression of the MC2 prodomain in mc2-1 results in constitutive activation of defense responses dependent on BAK1/BKK1 and SOBIR1, suggesting that the prodomain of MC2 specifically promotes immune signaling mediated by the receptor-like-protein (RLP)-type PAMP receptors. This is in stark contrast to most proteases, the functions of which are manifested by promoting substrate proteolysis. Our study uncovers a novel functional and mechanistic link bridging an evolutionarily conserved metacaspase and the regulation of plant immunity. 3.2 Introduction Plants have evolved a sophisticated innate immune system to detect and ward off diverse invading pathogens. Besides pre-formed structural, physical, and chemical barriers, plants have developed a multilayered inducible defense system. Known as PAMP-triggered immunity (PTI), the first line of inducible defense is launched upon recognition of the pathogen-derived conserved molecular signatures termed pathogen/microbe-associated molecular patterns (PAMPs or MAMPs) by plasma membrane-resident pattern-recognition receptors (PRRs). The vast majority of PRRs belong to the receptor-like kinase (RLK) and the receptor-like protein (RLP) families. To 57 overcome PTI, pathogenic microbes have evolved virulence machinery to deliver effector proteins to the plant apoplast or into host cells to subvert PTI signaling and manipulate host physiology to favor pathogen fitness. Plants, in turn, have developed resistance proteins (R proteins) to recognize pathogen effectors directly or indirectly, and thus mount effector-triggered immunity (ETI). Most R proteins belong to the class of intracellular nucleotide-binding leucine-rich repeat domain receptors (NLRs). However, immunity is a double-edged sword. While insufficient defense causes plant disease, excessive immunity typically leads to tremendous growth retardation, as overactivation of immune response is energetically and metabolically costly. One extreme example is the so-called autoimmune mutants, which mount defense constitutively and therefore normally exhibit dwarf morphology and sometimes display spontaneous cell death. Dozens of autoimmune mutants have been isolated in previous genetic screens, and their causative mutations are typically gain-of-function mutations in positive immune regulators such as NLRs, or loss-of-function mutations in negative regulators (van Wersch et al., 2016). Previously, our lab identified a novel autoimmune mutant named mc2-1, which contains a T-DNA insertion inside a metacaspase-encoding gene, called AtMC2 (AT4G25110). Metacaspases (MCs) are a group of cysteine proteases found in protozoa, fungi, and plants (Uren et al., 2000). In the MEROPS peptidase database, they belong to the C14 family of clan CD, together with caspases, paracaspases, and orthocaspases. These evolutionarily-related proteases feature a unique caspase-hemoglobinase tertiary fold and a conserved cysteine-histidine catalytic dyad (Uren et al., 2000). The signature proteases of this C14 family are metazoan caspases, which play essential roles in animal apoptotic pathways and inflammatory responses (Nagata, 2018). Similar to caspases, several plant MC genes have been described as important regulators of 58 programmed cell death (PCD) and stress responses (Tsiatsiani et al., 2011). This view is primarily based on phenotyping the MC loss-of-function mutants, although in most cases, the exact molecular mechanisms remain elusive. However, there are critical differences between MCs and caspases regarding the biochemical properties, suggesting MCs may have evolved a distinct mode of action (Klemencic and Funk, 2018a). One radical difference lies in their substrate specificity; while caspases perform cleavage after an aspartate, all three types of MCs prefer target sites with arginine or lysine at the P1 position (McLuskey et al., 2012; Vercammen et al., 2004). MCs are subdivided into three types based on the presence (type I) or absence (type II/III) of the N-terminal prodomain and the order of p20-p10 subunits. The Arabidopsis genome contains three type-I MC-encoding genes (AtMC1-AtMC3) and six type-II (AtMC4-AtMC9), which so far are the best characterized MCs in plants. Accumulating evidence suggests that plant MCs are pivotal players in defense responses. For instance, several type-II MCs, such as MC4, have been demonstrated to mediate the proteolysis-dependent maturation of endogenous DAMP peptides termed peps (Hander et al., 2019; Shen et al., 2019). A type-I MC, MC1, was shown to promote pathogen-induced cell death, a process that is negatively regulated by MC2 via a yet to be defined mechanism (Coll et al., 2010). However, whether MC2 is connected to immune regulation via MC1-independent pathways remains scarcely explored. In this project, we discovered a crucial role of MC2 in promoting PTI signaling mediated by the RLP-type PRRs by characterizing an autoimmune mc2 mutant allele called mc2-1. We demonstrate that the autoimmune phenotypes of mc2-1 are due to overexpression of MC2 prodomain rather than loss of MC2 function, suggesting an unprecedented proteolysis-independent mode of action of C14 proteases. 59 3.3 Materials and methods 3.3.1 Plant materials and growth condition All wild-type and mutant Arabidopsis plants are in the Columbia (Col-0) ecotype background. The original mc1 mc2 GABI-Kat mutant (TAIR Germplasm: CS2103350) was obtained from the GABI-DUPLO collection (https://www.gabi-kat.de/db/duplopair.php?pair_id=2565) and has been reported previously (Bolle et al., 2013). The mc1 (AT1G02170, GK-096A10) and mc2 (At4g25110, GK-537H06) single mutants were isolated from the F2 population of a cross between mc1 mc2 GABI-Kat allele and wild type Col-0, and for simplicity, referred to as mc1-1 and mc2-1 throughout this dissertation. An alternative mc1 allele, in which the entire MC1 coding sequence was deleted using the CRISPR-Cas9 system, was named mc1-2. Two additional T-DNA alleles of MC2, referred to as mc2-2 (WISCDSLOX357C10) and mc2-3 (SALK_009045) respectively, were obtained from the Arabidopsis Biological Resource Center at Ohio State University (ABRC). A deletion mutant of MC3, referred to as mc3-1, was also generated by CRISPR-Cas9-based genome editing. The bak1-4 (SALK_116202), bak1-5 bkk1-1, sobir1-12 (SALK_050715) and agb1-2 (CS6536), eds1-2 and pad4-1 mutants have been described previously (Joo et al., 2005; Kemmerling et al., 2007; Schwessinger et al., 2011; Wang et al., 2007). High-order mutants combining the above-mentioned mutant loci were generated by genetic crossing. All genomic loci were validated by PCR-based genotyping using specific primers listed in Table 3.1. Plants were grown on soil under short-day conditions (12 hrs light, 22 \u00E2\u0084\u0083; 12 hrs dark, 18 \u00E2\u0084\u0083) unless otherwise stated. Half-strength Murashige and Skoog (\u00C2\u00BD MS) medium-grown plants were cultivated inside a growth chamber under long-day conditions (16-hr light, 22 \u00E2\u0084\u0083/8-hr dark 19 \u00E2\u0084\u0083). 60 3.3.2 Molecular cloning and plasmid construction For expression of MC2 and other metacaspases, the full-length open reading frames (ORFs) of the target genes were amplified with Phusion DNA polymerase using primers listed in Table 3.1. The amplicons were then ligated into a modified pCambia1300 vector containing a cauliflower mosaic virus (CaMV) 35S promoter and C-terminal hemagglutinin (HA) tag sequence. The resulting plasmids were then transformed into Agrobacteria strain GV3101, before being introduced into the plant genome via floral dipping. For constructing the vector overexpressing the MC2 prodomain, the primer pair, Mc2CdsXhoIF /Mc2E1R3 (Table 3.1), was used to amplify a fragment coding for the MC2 prodomain with Phusion DNA polymerase (Thermal Scientific, Inc.). The resulting fragment was digested with XhoI/StuI and then ligated into a pCambia1300 vector that had been cut with SalI/StuI. The expression of the transgene was driven by a CaMV 35S promoter inside the pCambia1300 vector. The resulting constructs were introduced into wild type and suppressor line rom1-3 mc2-1 plants by floral dipping. For co-immunoprecipitation assays, the MC2 prodomain coding sequence and full-length MC2 ORF were amplified with primer pairs Mc2CdsXhoiF/Mc2StuiR2 and Mc2CdsXhoiF/Mc2E1R3, respectively. The PCR fragments were then inserted into a pCambia1300-3FLAG vector that had been digested by XhoI and StuI. To create constructs for generating metacaspase deletion mutants, CRISPR-Cas9 constructs expressing two guide RNAs targeting the desired metacaspase loci were constructed by replacing the default guide RNA sequences in the pHEE401 vector (Xing et al., 2014), using specific cloning primers listed in Table 3.1. The resulting CRISPR constructs were then introduced 61 into wild type plants. In the T1 generation, transgenic lines with presence of the desired deletion were identified by PCR-based genotyping. All constructs were confirmed by Sanger sequencing. 3.3.3 EMS Mutagenesis and mc2-1 suppressor screen EMS mutagenesis was carried out following a previously described protocol (Li and Zhang, 2016). In brief, the mc2-1 seeds were mutagenized in 20 mM ethyl methanesulfonate (EMS) for 16 hrs with gentle shaking. The mutagenized seeds were sterilized and plated on \u00C2\u00BD MS medium. Approximately 3,000 10-d-old M1 seedlings were grown to maturation, and their seeds were harvested in pools. In the next generation, approximately 60,000 M2 plants were screened for putative mc2-1 suppressors, which show increased plant size and reduced PR gene expression. Such putative suppressor lines were named rescuer of mc2-1 (rom) mutants. 3.3.4 Mapping by sequencing with backcross population To create backcross populations for whole-genome sequencing, homozygous mc2-1 suppressors lines were crossed with the parental mutant, mc1-1 mc2-1. In the F2 populations, only the F2 segregants showing clearly larger size were kept and selfed to generation an F3. Plant tissue from approximately 30 homozygous F2 lines was pooled together for DNA extraction. The resulting DNA samples were sequenced by Novogene (https://en.novogene.com/) using the Illumina HiSeq 2000 platform. Subsequent data processing and variant calling were performed following the GATK Best Practice workflow (McKenna et al., 2010). The frequency of the genuine SNPs was calculated with Excel to identify the linkage regions and candidate rom mutations. 62 3.3.5 Pathogen infection assays For bacterial infection assays, Pseudomonas syringae pv. maculicola (P.s.m.) strain ES4326, or specific P. syringae pv. tomato (P.s.t.) strains were hand-infiltrated into two fully-expanded leaves of four-week-old soil-grown plants. The dosage used for each infection experiment was noted in the corresponding figure legend. Infected leaves were sampled at day 0 or day 3 post-infiltration. During sampling, one leaf disc was punched from each leaf and two leaf discs from each plant were pooled as one sample. The samples were ground and diluted serially in 10 mM MgCl2, before being plated on LB plates supplemented with appropriate antibiotics. Bacterial colony-forming units (CFU) were quantified after incubation at 28\u00C2\u00B0C for 36 hrs. For Hyaloperonospora arabidopsidis (H.a.) Noco2 infection, two-week-old soil-grown Arabidopsis seedlings were sprayed with conidiospore solution at a dosage of 5\u00C3\u0097104 spore/ml. Plants were kept at 18 \u00E2\u0084\u0083 under a short-day light regime for seven days before the level of sporulation was quantified. 3.3.6 Gene expression analysis For regular gene expression analysis, 12-day-old plate-grown seedlings were directly collected for RNA extraction. For PAMP-induced gene expression, seedlings were thoroughly sprayed with 100 nM of the selected elicitors (flg22, nlp20, elf18) and harvested at 4 hrs after treatment. RNA was extracted using the EZ-10 Spin Column Plant RNA Mini-Preps Kit from Biobasic (Canada). Reverse transcription was carried out with the EasyScript\u00E2\u0084\u00A2 Reverse Transcriptase (ABM, Canada). Quantitative PCR (qPCR) was performed using the SYBR Premix Ex TaqII kit (Takara, Japan). Expression values were normalized to the expression of ACTIN1 and analyzed in Excel. Primers used for gene expression analysis were listed in Table 3.1. 63 3.3.7 Firefly luciferase complementation assay For luciferase complementation assay, Agrobacterium strain GV3101 carrying different luciferase constructs was grown overnight at 28 \u00E2\u0084\u0083 in LB broth containing proper antibiotics. Agrobacterium cells were harvested by centrifugation and diluted into OD600 = 0.3 with the resuspension buffer (10 mM MgCl2, 10 mM MES pH 5.4, and 200 \u00C2\u00B5M acetosyringone). Agrobacteria suspensions expressing the two fragments of luciferase were mixed according to the experimental design. Afterward, the agrobacteria mixtures were infiltrated into four- to five-week-old N. benthamiana leaves, 36 hrs after which 200 \u00C2\u00B5M D-Luciferin (Gold Biotechnology) was infiltrated into the same leaf sectors. The chemiluminescence signal was then detected with a bio-rad ChemiDoc XRS+ machine. 3.3.8 Membrane fractionation assay The procedure of membrane fractionation was modified from a previous report (LaMontagne et al., 2016). Briefly, 3 g of two-week-old transgenic Arabidopsis seedlings expressing hemagglutinin (HA)-tagged MC2 were collected and ground into fine powder in liquid nitrogen with a mortar and pestle. All the following steps were performed on ice or at 4 \u00E2\u0084\u0083. The tissue powder was dissolved and then homogenized with 6 ml of membrane isolation buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.3 M sucrose, 5 mM EDTA, 5 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 2 mM DTT, 1\u00C3\u0097 protease inhibitor cocktail from Roche) for 0.5 hr. After a small aliquot was saved as a sample of the total protein, the bulk cell lysate was spun down twice at 15,000 g for 15 min to sediment cell debris, thereby removing nuclei and some unwanted organelles such as mitochondria and lysosomes. Next, the supernatant was transferred to the ultracentrifuge tubes, 64 strictly balanced, and spun down at 100,000 g for 1 hr to sediment the membranes. The resulting supernatant and pellet were saved as the soluble fraction and Membrane fraction, respectively. All protein samples were homogenized in 2\u00C3\u0097SDS loading buffer and then subjected to immunoblotting with either an anti-HA or anti-BAK1 antibody. 3.3.9 Co-immunoprecipitation (Co-IP) assay Co-immunoprecipitation (Co-IP) in N. benthamiana was carried out as described previously with minor modification (Xu et al., 2015). Briefly, agrobacteria strains carrying constructs expressing different epitope-tagged proteins were infiltrated into N.B. leaves at a dosage of OD600 = 0.3. Two days post-infiltration, ~1.5 g of the infiltrated leaf tissue was sampled and ground into fine powder in liquid nitrogen with a mortar and pestle. All the followings steps were conducted either on ice or in a 4 \u00E2\u0084\u0083 cold room. To start with, each sample was resuspended with two volumes (2 ml per g) of extraction buffer (10% glycerol, 25 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.15% NP-40, 1mM NaF, 1mM PMSF, 10 mM DTT, 2% PVPP, 1\u00C3\u0097 protease inhibitor cocktail from Roche) and ground with pestle to achieve homogeneity. After removing cell debris via two rounds of centrifugation at 15,000 g, the cell lysate was incubated with anti-FLAG M2 beads (Sigma) for 2 hrs with gentle shaking. Following incubation, the M2 beads were collected by gentle spinning and washed three times with fresh extraction buffer, before the bead-bound protein was eluted with hot 1\u00C3\u0097SDS loading buffer. The eluted protein samples, together with the input samples, were analyzed by western blot. Table 3.1 Primers used in Chapter 3 65 Primer Name Sequence (5\u00E2\u0080\u0099 to 3\u00E2\u0080\u0099) Mc2SfiiF CGCGGATCCGGCCGTCAAGGCCATGTTGTTGCTGGTGGACTG Mc2SfiiR CGCGAATTCGGCCCATGAGGCCTAAAGAGAAGGGCTTCTCATATAC MC2 T-DNA F CAGAAGCGAGCGGTGATAGT MC2 T-DNA R ACGATGGCATGGAGCTTAAC Mc2RtR TCTTCTTCGGTGAGCATAAGAA Mc2CpcrF GATGAGGAAGAGGAAGTAAACC C256AF ATCGTCGACGCTGCT CATAGTGGTA C256AR TACCACTATGAGCAGCGTCGACGAT C135AF ACTCAAAGGAGCT ATCAATGACGC C135AR GCGTCATTGATAGCTCCTTTGAGT Mc2CdsXhoIF CCGCTCGAGATGTTGTTGCTGGTGGACTG Mc2BamhiR CGGGGATCCTAAAGAGAAGGGCTTCTCATATAC Mc2E1R3 GAAGGCCTGATACATCCTTTGAGTTCGTC Mc2SfiiF2 CGCGGATCCGGCCGTCAAGGCCA ATGTTGTTGCTGGTGGACTG Mc2CdXhoiF CCGCTCGAGGCGGTGATAGTCGGGGTTT Mc2StuIR GAAGGCCTTTATAAAGAGAAGGGCTTCTCATA Mc2StuiR2 GAAGGCCTTAAAGAGAAGGGCTTCTCATATAC Mc2E1BamhiR CGGGGATCCGTGTTTGAGTTCGTCCTTTG Gabi3144 GTGGATTGATGTGATATCTCC Mc1XhoiF CCGCTCGAGCCGATTCGTCTTCATCTGATT Mc1StuiR GAAGGCCTCTAGAGAGTGAAAGGCTTTG Mc1RtF ATGCTTACCGAGGAAGAAACT Mc1RtR CAGCTCTGTTCATTCTGCAT MC1 T-DNA F CTCTGCCAGGCTGTTACTCA 66 MC1 T-DNA R GAGCCAATACAATGCCATCC Mc1BsF ATATATGGTCTCGATTGATCTACGCTCCTCCGATGCGTT Mc1F0 TGATCTACGCTCCTCCGATGCGTTTTAGAGCTAGAAATAGC Mc1R0 AACTTACTAGAGAGTGAAAGGCCAATCTCTTAGTCGACTCTAC Mc1BsR ATTATTGGTCTCGAAACTTACTAGAGAGTGAAAGGCC Mc1DelF TCGACTGGAACGCAATAAGA Mc1DelR AAGTATGCAGCACCAGAAGT Mc1XhoiF CCGCTCGAGCCGATTCGTCTTCATCTGATT Mc1E1R GAAGGCCTTTAGGTAAGCATGAGAATTGAATC Mc3E1F CCGCTCGAGATGGCTAGTCGGAGAGAAG Mc3E1R GAAGGCCTTTATGTGAGCATGAGAATAGAGT Mc3SmaiR TCCCCCGGGTCAGAGTACAAACTTTGTCGC Mc3BsF ATATATGGTCTCGATTGAAGCTCAATCATGGCTAGTGTT Mc3F0 TGAAGCTCAATCATGGCTAGTGTTTTAGAGCTAGAAATAGC Mc3R0 AACAGCTATTTGCAGCATCATACAATCTCTTAGTCGACTCTAC Mc3BsR ATTATTGGTCTCGAAACAGCTATTTGCAGCATCATAC Mc3DeF CCACTAATCGTCTCACCTTA Mc3DeR AGAGAGTATGGCTATGTATGC Mc8XhoiF CCGctcgagATGGCGAAGAAAGCACTTTTG Mc8StuiR GAaggcctGTAGCATATAAATGGTTTATCAAC Mc9XhoiF CCGctcgagATGGATCAACAAGGGATGGT Mc9StuiR GAaggcctAGGTTGAGAAAGGAACGTC Mc4SaliF gatccGTCGACATGACGAAAAAGGCGGTGCT Mc4StuiR GAaggcctACAGATGAAAGGAGCGTTG 67 BIR1-Kpn1-F CGGGGTACC ATGATGATGGGTAGGTTAG BIR1-Sal1-R AGCACGCGTCGACACGAGCAACTATGAGCTCTTC Cpk28SfiiF ATCCGGCCGTCAAGGCCATGGGTGTCTGTTTCTCCGCC Cpk28SfiiR AATTCGGCCCATGAGGCCTCGAAGATTCCTGTGACCTG 3.4 Results 3.4.1 Metacaspase 2 (MC2) is predominantly expressed in leaf tissue and transcriptionally upregulated upon pathogen infection Previously, an autoimmune mutant named mc2 was isolated from a reverse genetic screen undertaken by our former lab members (Xu, 2017). This particular mc2 mutant contains a T-DNA insertion inside the first exon of the type-I metacaspase (MC) gene MC2 (AT4G25110). This mc2 T-DNA mutant exhibits dwarfed stature (Figure 3.1B) and constitutive expression of defense marker genes PATHOGENESIS-RELATED GENE 1 (PR1) and PR2 (Figure 3.3D, E). Intriguingly, two additional T-DNA alleles of MC2, WISCDSLOX357C10 and SALK_009045, have completely wild type-like morphology (Figure 3.1B) and defense gene expression (data not shown). For the purpose of clarity, we renamed the original autoimmune mc2 T-DNA from GABI-Kat collection as mc2-1, and the other two alleles as mc2-2 and mc2-3 respectively (section 3.3.1 for more details). The T-DNA insertion sites of these three mc2 alleles were marked in Figure 3.1A. Identification of the mc2-1 autoimmune allele was exciting because very little was known about the involvement of plant MCs in the regulation of immune signaling. The prospect of unveiling such a functional and mechanistic link prompted us to study MC2 in more depth. 68 As a typical type-I metacaspase, the MC2 protein is composed of an N-terminal prodomain (PD) that harbors a zinc-finger motif and several proline-rich repeats, followed by a catalytic domain consisting of a p20 and a p10 subunit (Figure 3.1A). When translated, the first exon of MC2 corresponds to its PD, and the following four exons encode its protease domain (Figure 3.1A). The signature Cys (C256)-His (H200) catalytic dyad is located inside the p20 catalytic domain. We first retrieved the MC2 expression profile in different tissue types and developmental stages from the public transcriptome database (Winter et al., 2007). The MC2 transcript appears to be rather abundant in the rosette and cauline leaves, low in roots and the 2nd internode, and scarce in seeds and flowers, indicating a leaf-prioritized expression pattern (Figure 3.1C). Public microarray data also suggest that MC2 transcript appears to be induced under certain biotic stresses. To substantiate the microarray result, we infiltrated wild-type Col-0 plants with a virulent bacterial pathogen strain, P. syringae pv. maculicola (P.s.m.) ES4326, and then measured MC2 transcript abundance at 12 hrs or 24 hrs post-infiltration (hpi). As shown in Figure 3.1D, the MC2 transcript was modestly elevated at 12 hpi and drastically heightened at 24 hpi, confirming the transcriptional up-regulation of MC2 upon pathogen infection. Of note is that previous chromatin immunoprecipitation-sequencing (ChIP-seq) analysis identified MC2 as a candidate target of SARD1, which is a master transcription factor governing the expression of diverse defense-related genes (Sun et al., 2015). This further supports the involvement of MC2 in immune signaling. 69 Figure 3.1 The phenotype of three metacaspase 2 (mc2) mutant alleles and the expression pattern of MC2. 70 (A) Schematic diagram showing the genomic DNA (gDNA) and protein structure of MC2. Black squares represent the exons of MC2. The right-angle arrow indicates the transcription initiation site. The two white vertical lines indicate the start and stop codons, respectively. (B) Morphology of wild type (WT) and three different MC2 T-DNA alleles. The photo was taken for four-week-old plants grown under short-day conditions. (C) Expression profile of MC2 in different plant tissue types and at different developmental stages from public microarray databases (Winter et al., 2007). (D) MC2 transcript abundance measured by quantitative PCR (qPCR) at different timepoints following infiltration of the bacteria strain Pseudomonas syringae pv. maculicola (P.s.m.) ES4326. The ACT1 (AT2G37620) was used as an internal control. Error bars represent the standard deviation (SD) of three technical replicates. The assay was done twice with similar results. 3.4.2 MC2 localizes to both plasma membrane and cytosolic space To investigate the subcellular localization of the MC2 protein, we first constructed a vector expressing the MC2 protein with a C-terminal GFP fusion (MC2-C-GFP) under the control of a CaMV 35S promoter. Similar to the non-tagged version (Xu, 2017), transformation of the MC2-C-GFP construct into the mc2-1 background successfully rescued its dwarfed morphology (Figure 3.2A), suggesting the MC2-GFP is functional and most likely has similar subcellular localization as native MC2. We then transiently expressed MC2-C-GFP vector in N. benthamiana and examined the fluorescence signal through confocal laser scanning microscopy (CLSM). We observed strong fluorescence from the periphery of epidermal cells as well as faint signals from the cytosolic space (Figure 3.2 B), suggesting dual localization of MC2 both at the plasma membrane and in the cytosol. To corroborate our observation in CLSM, we performed a subcellular fractionation assay with the Arabidopsis transgenic plants expressing C-terminal 71 hemagglutinin (HA)-tagged MC2 (MC2-C-HA) and then determined the MC2-C-HA protein levels in different cellular fractions. While the transmembrane control protein BAK1 was exclusively present in the membrane fraction, MC2-HA was detected in both the membrane and soluble fractions, further supporting a dual distribution of MC2 in both the plasma membrane and cytosol pools. Examining the MC2 protein sequence for potential post-translational modification identified two putative palmitoylation sites, Cys7 and Cys10, both located in the MC2 prodomain. Palmitoylation is a common type of reversible lipidation, involving the covalent attachment of a palmityol group to primarily cysteine residues of the substrate, thus enhancing the hydrophobicity of substrate proteins and their membrane association (Smotrys and Linder, 2004). In light of this, MC2 likely localizes to the plasma membrane following palmitoylation, while the unmodified form may stay in the cytosol. (A) Morphology of four-week-old soil-grown plants of WT, mc2-1 and two independent transgenic lines expressing MC2-C-GFP in the mc2-1 background. Scale bar = 1 cm. Figure 3.2 MC2 localizes to the plasma membrane and cytosolic space. 72 (B) A representative graph showing the fluorescence signal when transiently expressing the 35S::MC2-C-GFP vector in N. benthamiana. Scale bar = 5 \u00C2\u00B5m. (C) Immunoblotting analysis of hemagglutinin (HA)-tagged MC2 protein (MC2-HA) in the indicated cellular fractions derived from transgenic plants expressing MC2-HA under a CaMV 35S promoter. BAK1 is utilized as a plasma membrane control. The upper three panels were initially derived from the same western blot membrane and later cropped during picture processing. 3.4.3 Epistatic analysis of the mc2-1 autoimmune mutant To dissect the immune signaling activated in the mc2-1 autoimmune background, we carried out epistatic analysis by introducing loss-of-function mutations of several well-studied positive immune regulators into the mc2-1 background, followed by phenotyping the resulting high-order mutants (Xu, 2017). Essential candidates include BAK1 and BKK1, two co-receptors important for PAMP recognition (He et al., 2018); SOBIR1, an adapter RLK required for function of the RLP-type PRRs (Liebrand et al., 2013; Liebrand et al., 2014b); AGB1, the beta subunit of the plant heterotrimeric G protein complex (Liu et al., 2013b); EDS1 and PAD4, two putative lipase-like proteins essential for the TNL-mediated ETI (Wiermer et al., 2005). 3.4.3.1 Knocking out either BAK1 or BKK1 strongly suppresses the autoimmune phenotype of mc2-1 BAK1 and its close homolog BKK1 serve as essential coreceptors for the majority of PAMP receptors (He et al., 2018). To probe whether PTI is involved in the mc2-1 autoimmunity, we examined the morphology of soil-grown mc2-1 bak1-4 mutant and found that mc2-1 bak1-4 was clearly larger in size than mc2-1 (Figure 3.3A). Besides rescuing the dwarfism, bak1-4 also strongly suppressed other mc2-1 autoimmune hallmarks such as constitutive expression of PR1 73 (Figure 3.3D) and PR2 (Figure 3.3E) as well as enhanced disease resistance against oomycete Hyaloperonospora arabidopsidis (H.a.) NOCO2 (Figure 3.3F). Like bak1-4, bkk1-1 also strongly suppressed mc2-1 autoimmunity outputs (Figure 3.4 B, D, E, F). Combined mutation of BAK1 and BKK1 completely reverted the mc2-1 morphology and immune responses back to the wild-type level (Figure 3.3C, D, E, F). Taken together, these results demonstrate that activation of BAK1/BKK1-dependent, presumably membrane PRR-mediated immune signaling, is responsible for the mc2-1 autoimmune phenotypes. Figure 3.3 Knocking out either BAK1 or BKK1 strongly suppresses the autoimmune phenotypes of mc2-1.\u00CF\u00AE 74 (A, B, C) Morphology of representative four-week-old soil-grown plants of the indicated genotypes. Scale bars = 1 cm. (D, E) Quantification of PR1 (D) and PR2 (E) expression levels in WT, mc2-1, bak1-4 mc2-1, bkk1-1 mc2-1, and bak1-5 bkk1-1 mc2-1. Error bars indicate the SD of three technical replicates. Both (D) and (E) are representative datasets of three independent experiments. (E) Growth of Hyaloperonospora arabidopsidis (H.a.) NOCO2 on the indicated plant genotypes. Two-week-old soil-grown seedlings were sprayed with 105 spores/mL NOCO2 conidia. The sporulation of H.a. was then measured at 7 days post-inoculation (7 dpi). Data were presented as Mean \u00C2\u00B1 SD with six biological replicates. Different letters indicate statistical difference (p < 0.05, one-way ANOVA, N = 6). \u00CF\u00AE Figure 3.3D, E, F were adapted from Figure 2.5B, C, D of Xu, 2017, respectively. 3.4.3.2 Loss of SOBIR1 function fully rescues the autoimmunity of mc2-1 SOBIR1 is a crucial PTI regulator specifically required for the function of RLP-type PAMP receptors (Liebrand et al., 2014b). As shown in Figure 3.4, sobir1-12 completely suppressed mc2-1 autoimmune hallmarks such as dwarf stature (Figure 3.4A), the heightened expression of PR1 (Figure 3.4B) and PR2 (Figure 3.4C), as well as hyper-resistance against oomycete H.a. NOCO2 (Figure 4D). These data reveal that promiscuous activation of SOBIR1-dependent, presumably RLP-type PAMP receptor-mediated immune signaling accounts for the autoimmunity in mc2-1 background. 75 (A) Morphology of four-week-old soil-grown plants of WT, mc2-1, sobir1-12, and sobir1-12 mc2-1. Scale bar = 1 cm. (B, C) Expression level of PR1 (B) and PR2 (C) relative to ACT1 in the indicated plant genotypes determined by qPCR. Data are representative of three independent experiments. (D) Sporulation of H.a. NOCO2 on WT, mc2-1, sobir1-12, and sobir1-12 mc2-1 plants at 7 dpi. Statistical differences marked by different letters were determined by One-way ANOVA with six biological replicates (p < 0.05). \u00CF\u00AE Figure 3.4B, C, D were adapted from Figure 2.6B, C, D of Xu, 2017, respectively. 3.4.3.3 Results of additional candidate genes in the epistatic analysis The heterotrimeric G protein functions as a convergent signaling node downstream of multiple PAMP receptors (Liang et al., 2016; Liu et al., 2013b). In Arabidopsis, AGB1 encodes Figure 3.4 Loss-of-function mutation of SOBIR1 fully rescues the autoimmune phenotype of mc2-1.\u00CF\u00AE 76 the beta subunit of the G protein complex. The agb1-2 mc2-1 double mutant displays intermediate immune phenotypes compared to mc2-1 (Xu, 2017), suggesting the immune signaling in mc2-1 partially depends on the function of AGB1. EDS1 and PAD4 are two pivotal regulators downstream of TNL receptors. It was shown that eds1-2 or pad4-1 only marginally suppressed mc2-1 morphology and heightened PR gene expression (Xu, 2017), indicating ETI likely had a very minor contribution to the mc2-1 autoimmunity. 3.4.4 MC2 does not directly cleave BAK1 or BKK1 Since the autoimmunity of mc2-1 completely depends on BAK1 and both MC2 and BAK1 could localize to the plasma membrane, we hypothesized that MC2 may target BAK1/BKK1 for cleavage, thereby down-regulating the PRR signaling. To test this possibility, we compared BAK1 protein abundance between mc2-1 and wild type using a specific antibody against BAK1. To prevent positive transcriptional feedbacks caused by pre-activation of defense, we also included eds1-2 mc2-1 and pad4-1 mc2-1 double mutants for comparison. As shown in Figure 3.5A, introduction of the mc2-1 mutation into WT, eds1-2, or pad4-1, did not clearly increase the amount of BAK1 protein present in the background plants. Likewise, coexpression of MC2 with HA-tagged BAK1 in N. benthamiana leaves neither diminished the amount of full-length BAK1-HA nor boosted the accumulation of its cleavage products (Figure 3.5B). When investigating the potential physical association between BAK1/BKK1 and MC2 in split luciferase assay and co-immunoprecipitation (Co-IP), we failed to detect any positive interaction in either system (Figure 3.5C, D). If the hypothesis that MC2 facilitates the proteolysis of BAK1 holds true, overexpression of MC2 will restrict BAK1 and BKK1 protein accumulation, thereby causing defects in BAK1-77 dependent defense signaling. However, assessing typical PTI hallmarks in MC2-OE-2 and MC2-OE-10, two validated MC2 overexpression lines (Figure 3.5E), showed that MC2 overexpression lines were not compromised in flg22-induced FRK1 or WRKY29 transcriptional induction (Figure 3.5F, G), MAP kinase activation (Figure 3.5H), and reactive oxygen species (ROS) production (Figure 3.5I). An endogenous DAMP, pep23, also elicits BAK-dependent PTI. The pep23-triggered ROS generation was not attenuated in MC2 overexpression lines either (Figure 3.5J). Taken together, our data did not support the model that MC2 directly targets BAK1 and BKK1 for cleavage. 3.4.5 MC2 is unlikely to regulate immunity via directly cleaving SOBIR1 Our epistasis results demonstrate that SOBIR1 is also required for mc2-1-mediated immune signaling, so we explored the possibility that SOBIR1 is a proteolytic target of MC2. However, the presence of MC2 appeared not to diminish the amount of full-length C-terminally HA-tagged SOBIR1 (SOBIR1-HA) protein in N. benthamiana. Furthermore, we failed to observe any physical association between MC2 and SOBIR1 in both split luciferase assay and Co-IP in N. benthamiana (Figure 3.6B, C). If activation of MC2 hampers SOBIR1 protein stability, overexpression of MC2 would lead to attenuation of SOBIR1-dependent PTI signaling, for example, nlp20-triggered responses. So we examined the nlp20-induced PTI hallmarks in two independent MC2 overexpression lines. No compromise in nlp20-induced activation of MAP kinases (Figure 3.6D), production of ROS (Figure 3.6E), and transcriptional upregulation of PTI marker genes (Figure 3.6F, G) was observed in MC2 overexpression lines in comparison to the wild type, suggesting that it is unlikely that MC2 modulates immunity by promoting the proteolysis of SOBIR1. 78 79 (A) Immunoblotting of BAK1 protein in WT, mc2-1, eds1-2, eds1-2 mc2-1, pad4-1, pad4-1 mc2-1 plants using an anti-BAK1 antibody. (B) Western blot of full-length HA-tagged BAK1 (BAK1-HA) co-transformed with either empty vector (EV, left) or vector expressing MC2 in N. benthamiana. Agrobacteria strains carrying the indicated constructs were infiltrated into N. benthamiana leaves, two days after which the infiltrated leaf area was sampled for western blot analysis. The asterisk represents the full-length BAK1-HA protein, while the arrow and arrowhead mark distinct cleavage products. (C) Split luciferase assay to test the potential physical association between BAK1 and MC2. The right diagram shows the Agrobacteria combinations used to infiltrate the corresponding leaf sectors in the left graph. MC2CA refers to the protease-dead version of MC2. This assay was repeated twice with consistent results. (D) Co-immunoprecipitation assay between HA-tagged BKK1 (BKK1-HA) and FLAG-tagged MC2 (MC2-FLAG). The experiment was performed three times in total with similar results. (E) Quantification of MC2 transcript level in WT and three independent Arabidopsis transgenic lines expressing MC2 driven by a CaMV 35S promoter. (F, G) FRK1 (F) or WRKY29 (G) transcript abundance in Arabidopsis plants of the indicated genotypes at 0 hr or 4 hrs after 100 nM flg22 treatment. The experiments were performed twice and a representative result was shown for each gene. (H) Immunoblotting of phosphorylated MAP kinases (pMAPKs) in the indicated plant genotypes left untreated or treated with 100 nM flg22 for 15 minutes. (I, J) Measurement of flg22- (I) or pep23- (J) induced production of reactive oxygen species in the indicated genotypes. Different letters represent statistical difference determined by one-way ANOVA with at least six biological replicates (p < 0.05, N \u00E2\u0089\u00A5 6). Figure 3.5 MC2 does not target BAK1/BKK1 for cleavage. 80 (A) Western blot of full-length HA-tagged SOBIR1 (SOBIR1-HA) co-transformed with empty vector (EV, left) or construct expressing MC2 in N. benthamiana. The asterisk represents the full-length SOBIR1-HA protein, while the arrowhead represents its cleaved product. (B) Split luciferase assay to test the potential physical association between SOBIR1 and MC2. The right diagram shows the Agrobacteria combinations used to infiltrate the corresponding leaf Figure 3.6 MC2 likely does not directly cleave SOBIR1. 81 area in the left graph. MC2CA represents the protease-dead version of MC2. This assay was repeated twice with similar results. MEKK1 and MKK6 were included as a positive control. (C) Co-immunoprecipitation assay between SOBIR1-HA and MC2-FLAG using the transiently expressed proteins in N. benthamiana. The asterisk and the arrowhead represent full-length and degraded SOBIR-HA, respectively. The arrow marks an unspecific protein. (D) Western blot of phosphorylated MAP kinases (pMAPKs) in the indicated plant genotypes untreated or treated with 100 nM nlp20 for 10 min. (E) Measurement of nlp20-induced production of reactive oxygen species in the indicated genotypes. Different letters represent statistical differences determined by one-way ANOVA (p < 0.05, N \u00E2\u0089\u00A5 8). (F, G) Measurement of FRK1 (F) or WRKY29 (G) transcript abundance in the indicated genotypes at 0 hr or 4 hrs after 100 nM nlp20 treatment. 3.4.6 Characterization of mc2-1 suppressors In parallel with the aforementioned epistatic analysis, we conducted a suppressor screen in the mc2-1 background aiming to gain more insights into the MC2-regulated immune signaling (Xu, 2017). We used the mc2-1 background to produce an ethyl-methanesulfonate (EMS)-mutagenized population and screened for EMS-induced mutations that suppress the autoimmunity of mc2-1. Heritable mutations identified from the EMS screen were named Rescuers of mc2-1, or ROMs. For simplicity, we also assigned a unique lab code to each independent suppressor line. Ten putative suppressor lines were identified from the preliminary screen (Xu, 2017). Later we decided to focus on seven of them because these seven exhibited relatively stronger and more stable suppression. Compared to the mc2-1 background, the selected mc2-1 suppressor lines displayed markedly larger plant size (Figure 3.7A), substantially lower PR1 expression (Xu, 2017), and significantly reduced resistance against H.a. NOCO2 (Xu, 2017). Unexpectedly, the rom 82 mutations in these suppressors were all dominant or semi-dominant (Figure 3.8C), which is intriguing because dominant mutations are usually rare in EMS-based forward genetic screens. Morphology of five-week-old soil-grown plants of WT, mc2-1, and seven mc2-1 suppressor lines. Each suppressor line is labeled with a unique lab code shown underneath the corresponding plant. Scale bar = 1 cm. 3.4.7 Positional cloning of the rom mutations with mapping-by-sequencing We then decided to map the causative mc2-1 suppressor mutations, utilizing the mapping-by-sequencing technique established previously (James et al., 2013). First, we generated backcross populations for all suppressor lines by crossing them with the parent mc2-1. The F2 segregants Figure 3.7 Morphology of seven putative mc2-1 suppressor lines characterized in this study. 83 displaying clear suppressor phenotypes were selected. Due to the dominant nature of these rom mutations, F3 seeds from individual F2 lines were collected and planted to determine which lines are homozygous for the suppressor mutation. For four suppressor lines (3-17-2, 6-11-2, 6-15-2, 7-1-2), F3 seedling tissue from approximately 30 homozygous F2 lines was collected and subjected to genomic DNA extraction. The extracted DNA samples were then used for next-generation sequencing. Subsequent in silico analysis identified EMS-induced mutations across five chromosomes in these suppressors and the genome-wide distribution of the mutant allele frequency of all SNP loci was then plotted. Conceptually, the frequency of mutant SNPs unlinked to rom mutations is expected to fluctuate around 0.5 due to independent assortment; such a value will surge towards 1 for the SNPs in proximity to the rom loci due to genetic linkage. As shown in Figure 3.8A, the rom mutation in 6-15-2 was strongly linked to a 7-million-bp-long (8.12 Mb-15.23 Mb) region on chromosome 4 with around 10 candidate mutations. Subsequent fine-mapping analysis using SNP-based molecular markers further narrowed this rom mutation down to merely three candidates. Directed by a similar mapping procedure, the rom mutation of 7-1-2 was mapped to a region also in chromosome 4 with only four possible candidates. Strikingly, one locus, AT4G25110, was shared in the candidate lists of 6-15-2 and 7-1-2, indicating that these two suppressors were in fact allelic. This locus is, intriguingly, MC2, where the mc2-1 T-DNA is inserted. Subsequent sequencing analysis also revealed mutations in the MC2 open reading frame (ORF) in another four suppressor lines (Figure 3.8B, C). The only exception appeared to be 12-11-1, in which MC2 ORF was free of mutation. Hence, we re-named all the intragenic suppressors as rom1 with altogether four distinct alleles (rom1-1 to rom1-4), and the line 12-11-2 as rom2 (Figure 3.8B, C). 84 (A) Scatter plots showing the mutant SNP frequency of EMS-induced mutations across five chromosomes (Chrs) of the suppressor line 6-15-2. Each dot represents a distinct SNP locus Figure 3.8 The rom1 mutants are intragenic suppressors. 85 and the Y-axis indicates the frequency of the mutant allele. The black dot marks the causative mutation in 6-15-2 and the red dots represent other unrelated mutations. (B) The schematic structure of MC2 genomic DNA (gDNA) labeled with all intragenic suppressor mutations. Black squares represent the exons of MC2. Green lines mark the position of the four rom1 mutations. The magenta vertical line inside the first exon marks the position of mc2-1 T-DNA insertion. The right-angle arrow indicates the transcription initiation site. (C) Summary of the mc2-1 suppressor lines characterized in this thesis project. ORF: open reading frame. 3.4.8 Overexpression of the MC2 prodomain activates immunity To understand why rom1 mutation can rescue the mc2-1 autoimmunity, we first looked at the positions of the four rom1 alleles. Intriguingly, these four missense mutations are all contained in the first exon of MC2, which, synthesizes the prodomain (PD) when translated. Considering that the T-DNA insertion of mc2-1 resides at the end of the first exon, the PD segment can still be produced in the mc2-1 background. Using specific primers displayed in Figure 3.9A, we quantified the amount of the MC2-PD transcript, which represents the transcript segment coding for the prodomain, in wild type, mc2-1 and rom1-3 mc2-1. Strikingly, the MC2-PD transcript level in mc2-1 and mc2-1 rom1-3 are substantially higher than that of the wild type (Figure 3.9B). We then hypothesized that the heightened MC2-PD expression may account for the autoimmune phenotypes of mc2-1. To test this, we transformed one of the rom1 lines, mc2-1 rom1-3, with a vector expressing MC2-PD driven by a CaMV 35S promoter. Indeed, an appreciable fraction of the transformants phenocopied the mc2-1 parent plant, displaying dwarfed stature and dark-green leaves, indicating that the phenotypes of mc2-1 result from overexpression of the MC2-PD rather than loss of MC2 function. This finding is also consistent with the fact that two additional mc2 knockout alleles, mc2-2 and mc2-3, didn\u00E2\u0080\u0099t display autoimmune phenotypes. To further 86 corroborate our hypothesis, we introduced the 35S::MC2-PD construct into the wild type background and examined the growth of the resulting transformants. Similarly, overexpressing MC2-PD in wild type also leads to mc2-1-like dwarfism (Figure 3.9D), and constitutively activated defense as evidenced by heightened PR2 transcription (Figure 3.9E). It is interesting to note that, all four rom1 mutations cause substitution of proline residues inside the proline-rich repeats of the MC2 PD (Figure 3.8C), suggesting that these prolines are essential for the function of the MC2 PD in triggering defense. As shown in Figure 3.9B, the transcript of MC2-PD in rom2 mc2-1 was comparable to wild type, suggesting that the suppression of mc2-1 autoimmune phenotypes by rom2 is caused by restored expression of MC2-PD. 87 (A) Schematic representation of the first exon of MC2. Arrows labeled with \u00E2\u0080\u0098F\u00E2\u0080\u0099 and \u00E2\u0080\u0098R\u00E2\u0080\u0099 represent primers used in the quantitative PCR analysis of (B). The four light-green lines indicate the sites of four rom1 mutations. The black square represents the first exon of MC2 gDNA and the dashed ellipse represents the remaining part. This graph is not drawn to scale. (B) Quantitative PCR analysis of the amount of MC2-PD transcript in WT, mc2-1, and rom1-3 mc2-1 and rom2 mc2-1. MC2-PD transcript represents the transcript segment coding for the prodomain of MC2. The assay was repeated twice with similar results. (C) Morphology of four-week-old soil-grown plants of WT, mc2-1, and two independent transgenic lines expressing MC2-PD in the rom1-3 mc2-1 background driven by a CaMV 35S promoter. Scale bar = 1 cm. (D) Morphology of the three independent transgenic lines derived from wild-type plants transformed with 35S::MC2-PD. The photo was taken for three-week soil-grown plants under short-day conditions. Scale bar = 1 cm. (E) Measurement of PR2 expression level in three independent 35S::MC2-PD transgenic lines. Data are representative of two independent experiments and are shown as Mean \u00C2\u00B1 SD. 3.4.9 MC2 is unlikely to function by repressing type-II metacaspases In mammalian systems, the prodomains of initiator caspases serve as intrinsic inhibitory domains, which need to be removed to unleash the protease activity (MacKenzie and Clark, 2012; Shi, 2004). However, the biochemical function of the metacaspase prodomains remains unclear. One prevailing hypothesis is that the prodomains of MCs, similar to their counterparts in metazoan caspases, repress the activity of the protease domain by blocking the entry of proteolytic substrates (Klemencic and Funk, 2018a; McLuskey et al., 2012). This hypothesis prompted us to formulate a \u00E2\u0080\u0098type-II MC inhibitor\u00E2\u0080\u0099 model, in which, MC2 blocks the activity of certain type-II MCs via its prodomain, which acts as a protease inhibitor of the target type-II MCs. Figure 3.9 Elevated expression of MC2 prodomain (PD) leads to autoimmunity. 88 The Arabidopsis genome encodes six type-II MCs, which fall into three distinct monophyletic clades (Figure 3.10A). We selected one candidate from each clade, namely MC4, MC8, and MC9, to test our \u00E2\u0080\u0098type-II MC inhibitor\u00E2\u0080\u0099 model. These three type-II MCs are also expressed at relatively higher levels compared to other type-II MCs based on public microarray data. We first investigated the potential physical association between MC2 and the candidate type-II MCs via Co-IP. However, MC2 failed to interact with MC4 or MC8 in our Co-IP experiments (Figure 3.10B, C). We then sought for potential genetic evidence for the \u00E2\u0080\u0098type-II MC inhibitor\u00E2\u0080\u0099 model. Assuming the mc2-1 autoimmunity results from the inactivation of type-II MCs, overexpression of the target type-II MC into mc2-1 could potentially rescue its autoimmune phenotypes. So we transformed mc2-1 with vectors overexpressing MC4, MC8, and MC9, respectively, and examined the resulting transgenic population for potential suppression. However, all transgenic lines of the tested type-II MCs were indistinguishable from the background (Figure 3.E, F, G), although the MC proteins appeared to be abundant from the immunoblotting analysis (data not shown), suggesting that the mc2-1 autoimmunity was not caused by inactivation of MC4, MC8, or MC9. Furthermore, taking advantage of the CRISPR-Cas9 genome editing technique, we generated mc8 single, mc9 single, and mc4 mc5 mc6 mc7 quadruple mutants. None of these knockout mutants displayed mc2-1-like autoimmunity (data not shown). Taken together, these results suggest that MC2 probably does not fulfill its immune function through inhibiting MC4/MC8/MC9. 89 (A) Phylogenetic tree of the nine Arabidopsis metacaspase (MC) genes. The mouse (Mus musculus) Caspase 8 (CASP8) is included as an outgroup. This maximum-likelihood tree was generated using RAxML with GTRGAMMA models with the genomic DNA sequence. Numbers on top of branches indicate the bootstrap value out of 500 bootstrap practices. The three type-II MCs under test were colored in magenta. (B, C) Co-IP assay of MC2-FLAG co-expressed with MC4-HA (B) or MC8-HA (C) in N. benthamiana. MC4-HA or MC8-HA was transiently expressed in N. benthamiana leaves together with MC2-FLAG or an empty vector. Total protein was immunoprecipitated with anti-FLAG M2 agarose beads. Epitope-tagged proteins in the input and elution samples were detected using an anti-FLAG or anti-HA antibody. Figure 3.10 The selected type-II metacaspases did not physically associate with MC2 and failed to rescue mc2-1 autoimmune phenotypes. 90 (E, F, G) Morphology of WT, mc2-1, and three independent T1 transformants expressing MC4 (E), MC8 (F), or MC9 (G) under the control of 35S promoter. Scale bars = 1 cm. 3.4.10 Overexpression of MC1 or MC3 in mc2-1 strongly rescued its autoimmune phenotypes An alternative model we proposed to explain why overaccumulation of MC2-PD activates immunity, is termed a \u00E2\u0080\u0098dominant-negative effects\u00E2\u0080\u0099 model. Besides MC2, the Arabidopsis genome encodes two closely related type-I MCs, MC1 and MC3 (Figure 3.10A). We hypothesized that MC2-PD potentially bound to and interfered with the function of two other type-I MCs, which otherwise can compensate for the loss of MC2, thus causing dominant-negative effects. Oftentimes, dominant-negative effects occur owing to protein homo- or hetero-dimerization (or oligomerization) and the dysfunctional protein forms poisoned complex with other functional components (Veitia, 2007). We wondered whether full-length MC2 or its N-terminal prodomain fragment could interact with MC2 itself. We tested this idea with a co-immunoprecipitation assay and found that both full-length MC2-HA and MC2-PD-HA were clearly pulled down by MC2-FLAG (Figure 3.11E, F), suggesting self-association of MC2 via its prodomain. To test whether MC1 or MC3 can compensate for the loss of MC2, we transformed mc2-1 plants with constructs expressing C-terminally HA-tagged MC1 (MC1-HA) and MC3 (MC3-HA), respectively. Strikingly, introduction of MC1-HA or MC3-HA rescued the dwarfed morphology and constitutive PR2 expression of mc2-1 (Figure 3.11A, B, C, D), suggesting that MC1 and MC3 seem to be genetically equivalent to MC2. However, we could not exclude the possibility that the complementary effects from overexpressing MC1 or MC3 are caused by interference with the 91 excessive amount of MC2 prodomain in mc2-1. Given that there appeared to be prodomain-mediated self-association for type-I MCs (Figure 3.11E, F), it is possible that high levels of MC1/MC3 protein derived from the transgene binds to and inactivates the MC2 prodomain. (A, B) Morphology of four-to-five\u00E2\u0080\u0090week\u00E2\u0080\u0090old soil\u00E2\u0080\u0090grown plants of WT, mc2-1, and three independent transgenic lines expressing MC1 (A) or MC3 (B) in the mc2-1 background under a 35S promoter. Scale bar = 1 cm. Figure 3.11 Overexpression of MC1 or MC3 largely rescued the autoimmunity of mc2-1. 92 (C, D) Semi-quantitative PCR (Semi-qPCR) analysis of PR2 expression level in three independent transgenic lines expressing MC1 (C) or MC3 (D) in the mc2-1 background, together with WT and mc2-1. The ACTIN1 (ACT1) was used as a loading control. (E, F) Full-length MC2 and the MC2 prodomain (MC2-PD) physically interacts with MC2 in planta. MC2-HA (E) or MC2-PD-HA (F) was transiently expressed in N. benthamiana leaves together with MC2-3FLAG or empty vector. Total protein was immunoprecipitated with anti-FLAG M2 agarose beads. Epitope-tagged proteins in the input and elution samples were detected using an anti-FLAG or anti-HA antibody. (G) Morphology of four-week-old soil-grown plants of WT, mc2-1, and two mc1/2/3 triple mutant alleles. Scale bar = 1 cm. (H) PR2 expression of the plant genotypes in (G). Different letters represent statistical difference with three biological replicates (one-way ANOVA, p < 0.05, N = 3). Data are representative of three independent experiments. To further test this model, we created two versions of mc1/2/3 triple mutants via combining available T-DNA alleles or mutants containing CRISPR-Cas9-mediated deletion (section 3.3.1 for more details). The rationale was that if the \u00E2\u0080\u0098dominant-negative effects\u00E2\u0080\u0099 model holds true, the mc1/2/3 triple mutant will phenocopy mc2-1 or display even more pronounced autoimmunity. Surprisingly, the morphology of the two mc1/2/3 triple mutants was almost indistinguishable from the wild type (Figure 3.11G). In addition, in stark contrast to mc2-1, which displayed heightened PR2 expression, the mc1/2/3 triple mutants showed similar basal levels of PR2 transcript resembling wild type (Figure 3.11H). Consequently, the \u00E2\u0080\u0098dominant-negative effects\u00E2\u0080\u0099 model was rejected because of the discrepancy with the defense phenotype of mc1/2/3 triple mutants. 93 3.4.11 MC2 is required for basal immunity and nlp20-triggered PTI responses Since the prodomain has an immunity-stimulating activity, we then asked whether it was naturally produced as a potential means to boost defense signaling. Consistent with recent findings in Shen et al. (2019), our immunoblotting analysis revealed that in addition to the full-length MC2 zymogen, a lower band corresponding to the MC2 catalytic domain was clearly observed in Arabidopsis transgenic plants (Figure 3.12A) and N. benthamiana (data not shown), indicating natural release of the MC2 prodomain by active self-processing. Next, we sought to clarify the natural function of MC2 by characterizing its two genuine knockout mutants, mc2-2 and mc2-3. The mc2-2 T-DNA completely inactivates MC2 because the insertion resides within the prodomain; the mc2-3 is also a null allele probably due to either the absence of prodomain overexpression or usage of an inappropriate stop codon. As MC2-mediated immune signaling is SOBIR1-dependent, we examined the nlp20-induced responses rather than flg22 or elf18. We found that nlp20-induced upregulation of defense marker genes, FRK1 and WRKY29, was significantly compromised in mc2-2 and mc2-3 (Figure 3.12B, C), suggesting that MC2 is naturally required for full activation of PTI responses. In our infection assays, the two mc1/2/3 triple mutants were more susceptible to bacterial pathogens P.s.m. ES4326 (Figure 3.12D) and P.s.t. DC3000 (Figure 3.12E), and the mc2-3 single mutant was compromised in resistance against P.s.t. DC3000 (Figure 3.12E), suggesting MC2 is required for basal resistance. 94 Since there is no putative protein exportation signal peptide in the MC2 prodomain sequence (Data not shown), it is unlikely that the PD serves as an endogenous DAMP. An alternative hypothesis is that MC2 facilitates PTI signaling via autoprocessing-mediated production of its prodomain, which, once released, either blocks the activity of specific immune repressors or promotes the function of certain immune activators (Figure 3.12F). (A) Immunoblotting of the MC2 enzymogen (asterisk) and self-processed product (arrowhead) with two transgenic lines expressing HA-tagged MC2. FL, full-length; CD, catalytic domain. (B, C) Quantification of FRK1 (B) or WRKY29 (C) transcript levels of WT, sobir7-1, mc2-2 and mc2-3 untreated or treated with 100 nM nlp20 for 4 hrs. Data were presented as Mean \u00C2\u00B1 SD. Figure 3.12 MC2 is required for nlp20-induced immune responses and basal immunity. 95 Different letters represent statistical difference determined by one-way ANOVA (p < 0.05, N = 2). (D, E) Growth of P.s.m. ES4326 and P.s.t. DC3000 on WT, mc2-3 and two different mc1/2/3 triple mutants at 3 day post-infiltration (day 3). Different letters indicate statistical difference determined by one-way ANOVA (p < 0.05, N \u00E2\u0089\u00A5 6). Note: mc1/2/3-1 = mc1-2 mc2-3 mc3-1; mc1/2/3-2 = mc1-1 rom1-3 mc2-1 mc3-1. (F) Current working model depicting the mode of action of MC2 in immunity. MC2-Z, MC2 zymogen; MC2-PD, MC2 prodomain. Direct and indirect connections are not differentiated in this graph. 3.4.12 The MC2 prodomain associates with BIR1, but not CPK28, in the co-immunoprecipitation assay Negative regulation of the PAMP receptor complex has been extensively studied in recent decades (Couto and Zipfel, 2016). After a thorough search for known PTI negative regulators that potentially fit into our hypothesis, we decided to focus on BIR1, CPK28 for in-depth analysis owing to their plasma membrane localization and well-characterized genetic functions. BIR1 negatively regulates the RLP-type receptor complex by preventing the association between BAK1 and SOBIR1 (Liu et al., 2016). Remarkably, bir1-1, a knockout mutant of BIR1, also displays autoimmunity, which, similar to mc2-1, also depends on BAK1, SOBIR1, and partially AGB1 (Gao et al., 2009; Liu et al., 2013; Liu et al., 2016). CPK28, attenuates PTI signaling by affecting the protein stability of the RLCK BIK1 (Monaghan et al., 2014). We investigated the potential physical association between MC2-PD and BIR1 or CPK28 via co-immunoprecipitation assay in N. benthamiana. Strikingly, FLAG-tagged MC2-PD successfully pulled down BIR1 protein (Figure 3.13B), but not CPK28 (Figure 3.13A), suggesting a specific interaction between MC2-PD and BIR1. Considering phenotypic resemblance between 96 bir1-1 and mc2-1, the binding of MC2-PD with BIR1 likely blocks its repressor function leading to the formation and activation of the RLP-type PRR complex. (A, B) Co-IP analysis of the interaction between MC2 prodomain (MC2-PD) and BIR1 or CPK28. The 35S::CPK28 or 35S::BIR1 was transiently expressed in N. benthamiana leaves together with MC2-PD-3FLAG or an empty vector. Total protein was immunoprecipitated with anti-FLAG M2 agarose beads. Epitope-tagged proteins were detected with an anti-FLAG or anti-HA antibody. BIR1 protein was detected by a polyclonal anti-BIR1 antibody, whose specificity has been validated previously (Gao et al., 2009). 3.5 Discussion Metacaspases (MCs) have received substantial research interest ever since they were first identified two decades ago (Uren et al., 2000). Despite tremendous advances in studying their biochemical properties, potential defense-related roles of MCs and the mechanisms by which they function remain poorly understood. Here, by taking advantage of an autoimmune allele of MC2, we unraveled an immune function of this type-I MC in promoting the RLP-type-PRR-mediated immunity. Unlike other C14 family proteases, which function by promoting substrate proteolysis, the immune-stimulating activity of MC2 is mediated by its N-terminal prodomain. Through our Figure 3.13 The MC2 prodomain physically interacts with BIR1, but not CPK28 in planta. 97 forward genetic screen, we isolated several intragenic suppressors with mutations disrupting specific proline residuals inside the MC2 prodomain (Figure 3.8B, C). Further analysis revealed that the autoimmunity of mc2-1 was due to overexpression of the MC2 prodomain, rather than loss of MC2 function. 3.5.1 MC2 prodomain has an unprecedented immune-activating role Much research on metacaspases has been focused on the structure-function analysis. It is generally accepted that the p20 and p10 are the key catalytic units, altogether folding into a functional protease domain that contains the signature caspase-hemoglobinase tertiary fold and Cys-His catalytic dyad (Uren et al., 2000). A recent domain-swapping study revealed that specific Asp residues of the p20 subunit determine the Ca2+ dependency of type-II MCs (Fortin and Lam, 2018). However, the functional relevance of the prodomains of type-I MCs remains ill-defined. In the mammalian system, the prodomains of initiator caspases serve as intrinsic inhibitory modules of the protease domain and cleavage of the prodomain is a prerequisite for caspase maturation (Salvesen et al., 2016a). Likewise, the autoprocessing-mediated prodomain excision has also been documented for many type-I MCs (Asqui et al., 2018; Machado et al., 2013; McLuskey et al., 2012; Wong et al., 2012), but there is clear evidence that this process is not simply an analogous \u00E2\u0080\u0098release of repression\u00E2\u0080\u0099 step to the metazoan counterparts. For example, the unprocessed TbMC2 and Yca1 still retain their normal proteolytic activity against synthetic substrates in vitro, suggesting excision of prodomain may not be mandatory for TbMC2\u00E2\u0080\u0099s peptidase activity (Asqui et al., 2018; Gilio et al., 2017; Moss et al., 2007; Wong et al., 2012). Instead, accumulating evidence suggests that the prodomains can serve as regulatory modules by facilitating protein interaction and thus direct the metacaspases to the desired substrates. For 98 example, the prodomain of Yca1 was shown to mediate the delivery of Yca1 to insoluble protein aggregate to carry out its function (Lee et al., 2010). Hence, our results suggest that the MC2 prodomain appears to guide MC2 for membrane attachment and interaction with the downstream signaling components. Our results uncover that the prodomain of MC2 has a previously uncharacterized role in promoting defense responses. Overexpression of the MC2 prodomain activates immune signaling in a BAK1- and SOBIR1-dependent manner. Importantly, several proline residues located in the C-terminal proline-rich repeats are required for such a defense-promoting activity. These prolines are potential residues required for associating with the target immune regulators. Multiple alignment of the prodomain sequence from all three type-I MCs shows that the C-terminal sequence is strongly diversified and these proline sites are mostly non-conserved in MC1 and MC3, in contrast to a fairly conserved N-terminal zinc-finger region (Figure 3.14). Consistently, overexpressing the prodomain of MC1 or MC3 did not cause autoimmunity (data not shown), suggesting that the immunity-promoting activity appears to be unique to the MC2 prodomain. 99 The arrowheads indicate the proline residues of MC2 that were disrupted by the rom1 mutations. Interestingly, the prodomain also dictates MC2\u00E2\u0080\u0099s plasma membrane localization most likely through the two putative palmitoylation sites (Cys7 and Cys10). Such lipidation-dependent intracellular dynamic partitioning potentially allows for expanded functionalities, where the MC2 of membrane and cytosol pools may impact distinct target proteins and regulate pathways in distinct physiological and environmental contexts. Future site-directed mutagenesis analysis disrupting these two cysteine sites will help to clarify the functional significance of these palmitoylation sites. Previous studies suggest that MC2 plays a role in the negative regulation of cell death in ETI (Coll et al., 2010). Intriguingly, this function of MC2 is shown to be independent of its catalytic activity, as its catalytic-dead mutant successfully complemented the mc2 knockout mutant (Coll et al., 2010). Unlike the canonical cleavage-dependent mechanism, our studies unraveled a positive function of MC2 in PTI, intriguingly, also in a prodomain-mediated manner. Whether this cell death inhibition role is also facilitated by the MC2 prodomain remains to be determined in the future. It will also be interesting to investigate whether BAK1/BKK1 and SOBIR1 are also involved in the negative regulation of cell death in ETI. Figure 3.14 Multiple alignment of the prodomain sequence of three Arabidopsis type-I metacaspase by MAFFT (v7.452). 100 3.5.2 Working models illustrating the function of MC2 in plant immunity At present, how MC2 promotes plant immunity is not fully understood. The major gap lies in the understanding of how the prodomain activates the BAK1/BKK1- and SOBIR1-dependent defense signaling. Based on the finding in Xu, (2017) and Chapter 3 of the present thesis, two models are proposed and described as the following: 3.5.2.1 Releasing the repression of BIR1 by the MC2 prodomain The first model is illustrated in Figure 3.15A. In the absence of pathogen, MC2 zymogen (MC2-Z) is anchored to the plasma membrane due to palmitoylation at the two cysteine sites in the prodomain. At this point, BIR1 prevents the interaction between BAK1 and the RLP-SOBIR1 receptor module. Upon pathogen infection, the MC2-Z is activated presumably via Ca2+-dependent self-processing, thereby releasing the prodomain (PD) and the catalytic domain (CD) simultaneously. The self-cleavage of MC2 was clearly observed in the present study (Figure 3.12A). While the CD potentially stimulates yet to be defined downstream signaling events, the PD binds to BIR1 and thus inhibits its repressor activity, leading to the ligand-dependent formation of BAK1-RLP(s)-SOBIR1 tripartite complexes. Activated PAMP receptor complex then initiates cytoplasmic pathways involving phosphorylation of MAP kinases, which in turn switch on the activity of downstream transcription factors responsible for the expression of defense-related genes such as FRK1 and WRKY29. Undoubtedly, the validity of this model relies heavily on concrete physical interaction data between MC2-PD and BIR1. We have shown that MC2-PD can associate with BIR1 in the Co-IP assay in the N. benthamiana transient expression system (Figure 3.13B). Future research will then be focused on confirming the MC2-PD-BIR1 interaction using additional techniques such as 101 firefly luciferase complementation assay and TurboID-mediated proximity labeling (Trinkle-Mulcahy, 2019). It will also be interesting to investigate the dynamics of this association by comparing the interaction strength before and after pathogen infection. 3.5.2.2 Promoting the activity of BAK1/BKK1 or SOBIR1 by MC2 prodomain The second model is summarized in Figure 3.15B. Similar to the first model, MC2 is activated via Ca2+-mediated autoprocessing upon pathogen detection, thereby releasing the MC2 prodomain from its zymogen. The prodomain then turns on the PTI signaling by facilitating the function of coreceptors BAK1/BKK1 (Figure 3.15, arrow \u00E2\u0080\u00981\u00E2\u0080\u0099) or the adapter RLK SOBIR1 (arrow \u00E2\u0080\u00982\u00E2\u0080\u0099) or some other positive immune regulators. Such activation could be achieved via promoting the kinase activity, or stabilizing the active form of BAK1 or SOBIR1; alternatively, the prodomain may serve as a molecular bridge that connects BAK1 and SOBIR1 for heteromerization. Again, the model needs to be validated by multiple protein-protein interaction methods to confirm the binding of MC2-PD to BAK1 or SOBIR1. Once the physical interaction is verified, downstream biochemical assays will be conducted to determine the outcome of this physical interaction. 102 Upon infection, MC2 is activated through Ca2+-dependent auto-processing, leading to the release of its prodomain. The prodomain then activates defense signaling via potentially blocking the repressor activity of BIR1 (A) or promoting the function of BAK1 or SOBIR1 (B). Figure 3.15 Hypothetical models illustrating how MC2 modulates plant immunity. 103 3.5.3 Overexpression of MC2 prodomain in the mc2-1 mutant likely results from promiscuous enhancer elements inside the T-DNA The mc2-1 allele exhibits autoimmunity owing to promiscuous transcription of the MC2 prodomain coding sequence. Such a heightened MC2-PD transcript level most likely is not caused by positive transcriptional feedbacks because the rom1-3 mc2-1 had a comparable amount of MC2-PD expression to mc2-1 despite the absence of autoimmunity (Figure 3.9B). Instead, the increased MC2-PD expression is probably due to the influence of the 35S promoter located in the mc2-1 T-DNA. Historically, the 35S promoter inside the T-DNA has been reported to enhance the expression level and disrupt the tissue specificity of adjacent genes (Gudynaite-Savitch et al., 2009; Weigel et al., 2000; Yoo et al., 2005). In mc2-1, the native MC2 promoter likely borrows the enhancer element from the mc2-1 T-DNA to overexpress the MC2 prodomain. 3.5.4 Potential self-association-mediated functional control of Arabidopsis type-I metacaspases Yeast and protozoan metacaspase are believed not to form dimers as metazoan caspases do because crystallization of Yca1 and TbMC2 revealed a structure that precludes dimerization (Klemencic and Funk, 2019; Salvesen et al., 2016b). However, to date, no such crystal structure is available for plant MCs. Our Co-IP assay revealed clear prodomain-dependent self-association of MC2 in the N. benthamiana transient expression system (Figure 3.11E, F). Certainly, this interaction needs to be further confirmed in Arabidopsis with multiple protein-protein interaction assays. If the self-association occurs naturally in Arabidopsis, the biological meaning of this interaction is more likely to be inactivating the prodomain, considering that overexpression of full-length MC2 into mc2-1 rescues its autoimmunity (Xu, 2017), rather than serving as a step of 104 protease maturation as seen for caspases. The zinc-finger region may serve as a possible interaction platform for MC2 self-binding. Interestingly, MC1 was also reported to interact with itself and its prodomain segment in yeast two-hybrid (Coll et al., 2010). The functional relevance of plant type-I MC self-association awaits more clarification in the future. Lacking a prodomain, type-II metacaspases are mainly controlled by Ca2+-dependent self-cleavage at the linker region (Vercammen et al., 2004). Certain type-II MCs are also regulated by nitrosylation of catalytic sites, although the generality and in vivo relevance of this process remain ill-defined (Belenghi et al., 2007). Our protein-protein interaction analysis showed that there was no direct interaction between MC2 and MC4 or MC8 (Figure 3.10B, C), suggesting the function of MC4/MC8 was not directly orchestrated by MC2. Remarkably, recent publications revealed the importance of four type-II MCs (MC4-MC7) in amplifying defense signaling by releasing the conserved elicitor-active peptides termed peps from their respective precursors (Hander et al., 2019; Shen et al., 2019). It is very interesting that these structurally-related proteases have been tailored for specific roles in the regulation of biotic stress via targeting different components in the immune signaling network. These highly-conserved metacaspase genes can potentially be harnessed as powerful genetic materials to engineer broad-spectrum resistance in crops. 105 Chapter 4: Conclusions and future perspectives 4.1 Conclusions Climate change and plant disease epidemics impose tremendous pressure on global food production. Current disease management relies heavily on the application of synthetic chemical pesticides, which, however, incurs serious problems such as pest resistance, damage of natural habitats, and health risks on humans (Gupta and Dikshit, 2009). Urgent changes are needed in the coming decades in order to sustain global agricultural productivity while maintaining the well-being of natural habitats. Exploiting natural plant defense capacity by harnessing the molecular machinery governing plant innate immunity offers powerful and sustainable disease control strategies in the decades to come. Upon detecting the highly conserved PAMP molecules, the pattern recognition receptor (PRR) complex activates PAMP-triggered immunity (PTI) and thus provides broad-spectrum defense against diverse pathogens. Finetuning the behavior of the PRR complex is vital for the well-being of plant life, as the perturbation of components of the PRR complex typically causes disastrous outcomes on plant growth and defense (Domnguez-Ferreras et al., 2015; Roux et al., 2011a). The blueprint of how plants achieve such tight regulation on the PRR complex remains incomplete, although mechanisms such as transcriptional control, protein dephosphorylation, and prevention of receptor heteromerization have been previously reported (Couto and Zipfel, 2016). To further expand our knowledge in this field, my thesis projects are focused on the identification and functional studies of novel mechanisms that contribute to the intricate regulation of the PRR signaling. In the second chapter, I sought to clarify the functional significance of the BAK1 carboxyl-terminal tail (CT) region in distinct BAK1-mediated signaling pathways. Although the presence 106 of the BAK1 CT has been documented for many years, its role remains largely mysterious. Through phenotyping of a BAK1 CT-deletion mutant, sobir7-1, I discovered that the CT is required for BAK1\u00E2\u0080\u0099s function in PAMP-triggered immunity but is dispensable for cell death inhibition and brassinosteroid-mediated plant growth. The sobir7-1 mutant was severely compromised in major PTI hallmarks including activation of MAP kinases, generation of reactive oxygen species, and transcriptional upregulation of PTI marker genes. Subsequent mechanistic studies uncovered that CT deletion strongly impaired BAK1 autophosphorylation level, indicating that the BAK1 CT promotes its kinase activity. Bioinformatic analysis revealed that a large number of leucine-rich repeat receptor-like kinases (LRR-RLKs) contain a CT extension outside of their kinase domain, suggesting that the CTs may have evolved a general function to modulate activities of various LRR-RLKs. The third chapter of my thesis focuses on the functional characterization of MC2, a type-I metacaspase, in the regulation of immune signaling. Previously, MC2 was shown to negatively impact the cell death execution mediated by its close homolog MC1 during incompatible host-pathogen interactions (Coll et al., 2010). Through genetic analysis of an mc2 autoimmune allele, we uncovered a previously unknown function of MC2 in promoting PTI signaling specifically mediated by the receptor-like protein (RLP)-type PAMP receptors. The gain-of-function mutant of MC2, mc2-1, displays autoimmune phenotypes that are completely dependent on the PAMP coreceptor BAK1 and the adapter RLK SOBIR1. In contrast, loss of MC2 function leads to compromised PTI responses. We showed that MC2 underwent autoprocessing to release its immunogenic prodomain, which was believed to activate defense signaling via modulating the function of its target immune regulators. Taken together, our findings of this chapter illustrate a 107 new paradigm of an evolutionarily conserved metacaspase contributing to the PRR signaling via its unique defense-promoting prodomain. In summary, our studies shed new light on the complicated regulatory network of the function of the PAMP receptor complex. We uncover that the CT extension of the coreceptor BAK1 serves as an intrinsic modulator of its kinase activity and itself is differentially required for BAK1\u00E2\u0080\u0099s function in immune and developmental pathways. Our data also reveal an uncanonical protease-independent mechanism by which MC2 positively regulates PTI responses mediated by the RLP-type PRRs. 4.2 Future perspectives 4.2.1 Regulation of BAK1 function by the CT extension In Chapter 2, we provide compelling evidence showing that the BAK1 CT is differentially required for immune and developmental pathways. It was recently reported that four in vivo phosphosites inside BAK1 CT were vital for PTI signaling but trivial for BR-mediated plant growth (Perraki et al., 2018). Loss of these phosphosites, similar to deletion of CT, also strongly diminished BAK1 autophosphorylation, suggesting that these phosphosites are essential for the role of BAK1 CT (Perraki et al., 2018). However, how the CT modulates the kinase activity of BAK1 is currently unclear. Future crystal structure analysis of the BAK1 full-length protein should provide new insights to unlock this puzzle. Our bioinformatics analysis suggests that the CT extension is broadly present throughout the LRR-RLK family. Further studies are needed to investigate the functional relevance of CTs in other LRR-RLKs. For example, complementation tests can be conducted by transforming the CT-deletion mutants of the candidate LRR-RLKs into their respective knockout backgrounds to 108 determine the functional requirement of the CTs. Since the CTs of BAK1, BRI1 and mammalian receptor tyrosine kinases all appear to affect their kinase activity (Schlessinger, 2000; Wang et al., 2005; Wu et al., 2018), in vitro kinase assays should be carried out to determine the impact of the CTs on the kinase activity of the candidate LRR-RLKs. Moreover, in planta kinase activity profiling should be conducted to confirm the influence of CTs on the LRR-RLKs in phosphorylating their known substrates in vivo. Collectively, these proposed experiments will further advance our understanding of the generality and functional significance of CTs for a wide array of LRR-RLKs. 4.2.2 Regulation of PAMP-triggered immunity by MC2 Plant metacaspases have emerged in the past decades as pivotal players in stress responses. The Arabidopsis MC1 was defined as an essential executioner of hypersensitive cell death (Coll et al., 2010). MC9 was proposed to cleave a secreted protein GRI and thus release the elicitor-active peptide to trigger ROS-dependent cell death (Wrzaczek et al., 2015). MC4 and its close homologs were recently demonstrated to mediate the maturation of endogenous DAMP peptides known as peps (Hander et al., 2019; Shen et al., 2019). In Chapter 3, we uncovered an important role of MC2 in the RLP-type PRR-mediated immune signaling. The mc2 knockout mutants were compromised in nlp20-induced PTI marker gene expression as well as in basal immunity. To determine which signaling branches are governed by MC2, future research will be concentrated on in-depth phenotyping of the mc2 null alleles for other PTI hallmarks such as MAP kinase activation and ROS generation and growth of P.s.t. DC3000 hrcC. It is also interesting to compare distinct PAMPs in future PTI assays to ascertain whether the PTI defects of mc2 knockout mutants are specific to the RLP-type PRRs-mediated pathway. 109 The primary focus of future research is to reveal the mechanism by which the MC2 prodomain activates immunity. Currently, BIR1 stands out as a prominent target of MC2 prodomain, not only because both are involved in the BAK1- and SOBIR1-dependent immune signaling, but also because BIR1 could associate with MC2 prodomain physically in planta (Figure 3.13). In the future, this physical interaction will be corroborated by additional protein-protein interaction assays such as split luciferase and TurboID-mediated proximity labeling. Since disruption of particular proline residues in four rom1 alleles could suppress the mc2-1 autoimmunity, it will be interesting to conduct site-directed mutagenesis to ascertain whether mutations of these prolines impair the association between BIR1 and MC2 prodomain. Moreover, the connection between BIR1 and MC2 can be further tested genetically by overpressing BIR1 in the mc2-1 background to determine whether overaccumulation of BIR1 could relieve the inhibitory effects of MC2 prodomain. If the physical interaction between MC2 prodomain and BIR1 is concrete, the future investigation will focus on clarifying the biological meaning of this interaction. Genetically, the binding of the MC2 prodomain to BIR1 should abrogate its repressor function. One possibility, considering that the BIR1 kinase activity is crucial for its repressor activity, is that the MC2 prodomain binds to BIR1 and blocks its kinase activity (Gao et al., 2009). This can be tested in vitro by kinase assays with purified proteins, and in vivo by comparing BIR1 phosphorylation status in the wild-type and transgenic plants overexpressing the MC2 prodomain. Alternatively, MC2 prodomain releases BAK1 from the sequestration of BIR1 through physical binding competition, which can be tested in vivo through competition assay using techniques such as bimolecular fluorescence complementation or split luciferase. 110 Possible direct interaction between MC2 prodomain and BAK1 or SOBIR1 will also be investigated by multiple protein-protein interaction techniques. Some follow-up biochemical experiments such as in vitro kinase assay will be designed according to the results of the protein interaction assays. Overall, my thesis projects provide valuable insights into the understanding of the complicated regulatory network of PRR signaling in Arabidopsis. Due to the high-degree conservation of SERK proteins and metacaspases across diverse plant lineages, these findings of Arabidopsis genes will serve as imperative references for the functional studies of their orthologues in various crops. These critical PTI regulators can potentially be utilized as powerful tools to engineer durable and broad-spectrum disease resistance in crops in the future. .111 Bibliography Aan den Toorn, M., Albrecht, C., and de Vries, S. (2015). On the Origin of SERKs: Bioinformatics Analysis of the Somatic Embryogenesis Receptor Kinases. Mol Plant 8:762-782. Albert, I., Bohm, H., Albert, M., Feiler, C.E., Imkampe, J., Wallmeroth, N., Brancato, C., Raaymakers, T.M., Oome, S., Zhang, H., et al. (2015). An RLP23-SOBIR1-BAK1 complex mediates NLP-triggered immunity. Nat Plants 1:15140. Asai, T., Tena, G., Plotnikova, J., Willmann, M.R., Chiu, W.L., Gomez-Gomez, L., Boller, T., Ausubel, F.M., and Sheen, J. (2002). MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415:977-983. Asqui, S.L., Vercammen, D., Serrano, I., Valls, M., Rivas, S., Van Breusegem, F., Conlon, F.L., Dangl, J.L., and Coll, N.S. (2018). AtSERPIN1 is an inhibitor of the metacaspase AtMC1-mediated cell death and autocatalytic processing in planta. New Phytologist 218:1156-1166. Belenghi, B., Romero-Puertas, M.C., Vercammen, D., Brackenier, A., Inze, D., Delledonne, M., and Van Breusegem, F. (2007). Metacaspase activity of Arabidopsis thaliana is regulated by S-nitrosylation of a critical cysteine residue. Journal of Biological Chemistry 282:1352-1358. Bi, G.Z., Zhou, Z.Y., Wang, W.B., Li, L., Rao, S.F., Wu, Y., Zhang, X.J., Menke, F.L.H., Chen, S., and Zhou, J.M. (2018). Receptor-Like Cytoplasmic Kinases Directly Link Diverse Pattern Recognition Receptors to the Activation of Mitogen-Activated Protein Kinase Cascades in Arabidopsis. Plant Cell 30:1543-1561. Bigeard, J., Colcombet, J., and Hirt, H. (2015). Signaling Mechanisms in Pattern-Triggered Immunity (PTI). Molecular Plant 8:521-539. Bolle, C., Huep, G., Kleinbolting, N., Haberer, G., Mayer, K., Leister, D., and Weisshaar, B. (2013). GABI-DUPLO: a collection of double mutants to overcome genetic redundancy in Arabidopsis thaliana. Plant Journal 75:157-171. Boudsocq, M., Willmann, M.R., McCormack, M., Lee, H., Shan, L.B., He, P., Bush, J., Cheng, S.H., and Sheen, J. (2010). Differential innate immune signalling via Ca2+ sensor protein kinases. Nature 464:418-U116. Bozhkov, P.V., Suarez, M.F., Filonova, L.H., Daniel, G., Zamyatnin, A.A., Jr., Rodriguez-Nieto, S., Zhivotovsky, B., and Smertenko, A. (2005). Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis. Proc Natl Acad Sci U S A 102:14463-14468. Bradley, D.J., Kjellbom, P., and Lamb, C.J. (1992). Elicitor-Induced and Wound-Induced Oxidative Cross-Linking of a Proline-Rich Plant-Cell Wall Protein - a Novel, Rapid Defense Response. Cell 70:21-30. Cao, H., Glazebrook, J., Clarke, J.D., Volko, S., and Dong, X.N. (1997). The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 88:57-63. Catanzariti, A.M., Dodds, P.N., Ve, T., Kobe, B., Ellis, J.G., and Staskawicz, B.J. (2010). The AvrM Effector from Flax Rust Has a Structured C-Terminal Domain and Interacts Directly with the M Resistance Protein. Mol Plant Microbe In 23:49-57. 112 Cesari, S. (2018). Multiple strategies for pathogen perception by plant immune receptors. New Phytol 219:17-24. Cesari, S., Kanzaki, H., Fujiwara, T., Bernoux, M., Chalvon, V., Kawano, Y., Shimamoto, K., Dodds, P., Terauchi, R., and Kroj, T. (2014). The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance. Embo J 33:1941-1959. Chinchilla, D., Bauer, Z., Regenass, M., Boller, T., and Felix, G. (2006). The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception. Plant Cell 18:465-476. Chinchilla, D., Zipfel, C., Robatzek, S., Kemmerling, B., Nurnberger, T., Jones, J.D.G., Felix, G., and Boller, T. (2007). A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448:497-U412. Chisholm, S.T., Coaker, G., Day, B., and Staskawicz, B.J. (2006). Host-microbe interactions: shaping the evolution of the plant immune response. Cell 124:803-814. Chung, E.H., da Cunha, L., Wu, A.J., Gao, Z.Y., Cherkis, K., Afzal, A.J., Mackey, D., and Dangl, J.L. (2011). Specific Threonine Phosphorylation of a Host Target by Two Unrelated Type III Effectors Activates a Host Innate Immune Receptor in Plants. Cell Host Microbe 9:125-136. Coll, N.S., Smidler, A., Puigvert, M., Popa, C., Valls, M., and Dangl, J.L. (2014). The plant metacaspase AtMC1 in pathogen-triggered programmed cell death and aging: functional linkage with autophagy. Cell Death and Differentiation 21:1399-1408. Coll, N.S., Vercammen, D., Smidler, A., Clover, C., Van Breusegem, F., Dangl, J.L., and Epple, P. (2010). Arabidopsis Type I Metacaspases Control Cell Death. Science 330:1393-1397. Couto, D., and Zipfel, C. (2016). Regulation of pattern recognition receptor signalling in plants. Nat Rev Immunol 16:537-552. Day, B., Dahlbeck, D., Huang, J., Chisholm, S.T., Li, D.H., and Staskawicz, B.J. (2005). Molecular basis for the RIN4 negative regulation of RPS2 disease resistance. Plant Cell 17:1292-1305. de Oliveira, M.V.V., Xu, G.Y., Li, B., Vespoli, L.D., Meng, X.Z., Chen, X., Yu, X., de Souza, S.A., Intorne, A.C., Manhaes, A.M.E.D., et al. (2016). Specific control of Arabidopsis BAK1/SERK4-regulated cell death by protein glycosylation. Nature Plants 2. Devendrakumar, K.T., Li, X., and Zhang, Y.L. (2018). MAP kinase signalling: interplays between plant PAMP- and effector-triggered immunity. Cell Mol Life Sci 75:2981-2989. Ding, Y.L., Sun, T.J., Ao, K., Peng, Y.J., Zhang, Y.X., Li, X., and Zhang, Y.L. (2018). Opposite Roles of Salicylic Acid Receptors NPR1 and NPR3/NPR4 in Transcriptional Regulation of Plant Immunity. Cell 173:1454-+. Djamei, A., Schipper, K., Rabe, F., Ghosh, A., Vincon, V., Kahnt, J., Osorio, S., Tohge, T., Fernie, A.R., Feussner, I., et al. (2011). Metabolic priming by a secreted fungal effector. Nature 478:395-+. Dodds, P.N., Lawrence, G.J., Catanzariti, A.M., Teh, T., Wang, C.I., Ayliffe, M.A., Kobe, B., and Ellis, J.G. (2006). Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes. Proc Natl Acad Sci U S A 103:8888-8893. Domnguez-Ferreras, A., Kiss-Papp, M., Jehle, A.K., Felix, G., and Chinchilla, D. (2015). An Overdose of the Arabidopsis Coreceptor BRASSINOSTEROID INSENSITIVE1-113 ASSOCIATED RECEPTOR KINASE1 or Its Ectodomain Causes Autoimmunity in a SUPPRESSOR OF BIR1-1-Dependent Manner. Plant Physiology 168:1106-+. Du, J.B., Gao, Y., Zhan, Y.Y., Zhang, S.S., Wu, Y.J., Xiao, Y., Zou, B., He, K., Gou, X.P., Li, G.J., et al. (2016). Nucleocytoplasmic trafficking is essential for BAK1-and BKK1-mediated cell-death control. Plant Journal 85:520-531. Dubiella, U., Seybold, H., Durian, G., Komander, E., Lassig, R., Witte, C.P., Schulze, W.X., and Romeis, T. (2013). Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation. P Natl Acad Sci USA 110:8744-8749. Escamez, S., Andre, D., Zhang, B., Bollhoner, B., Pesquet, E., and Tuominen, H. (2016). METACASPASE9 modulates autophagy to confine cell death to the target cells during Arabidopsis vascular xylem differentiation. Biol Open 5:122-129. Fortin, J., and Lam, E. (2018). Domain swap between two type-II metacaspases defines key elements for their biochemical properties. Plant Journal 96:921-936. Frey, N.F.D., Garcia, A.V., Bigeard, J., Zaag, R., Bueso, E., Garmier, M., Pateyron, S., de Tauzia-Moreau, M.L., Brunaud, V., Balzergue, S., et al. (2014). Functional analysis of Arabidopsis immune-related MAPKs uncovers a role for MPK3 as negative regulator of inducible defences. Genome Biology 15. Fritz-Laylin, L.K., Krishnamurthy, N., Tor, M., Sjolander, K.V., and Jones, J.D.G. (2005). Phylogenomic analysis of the receptor-like proteins of rice and arabidopsis. Plant Physiology 138:611-623. Gao, M.H., Wang, X., Wang, D.M., Xu, F., Ding, X.J., Zhang, Z.B., Bi, D.L., Cheng, Y.T., Chen, S., Li, X., et al. (2009). Regulation of Cell Death and Innate Immunity by Two Receptor-like Kinases in Arabidopsis. Cell Host Microbe 6:34-44. Gilio, J.M., Marcondes, M.F., Ferrari, D., Juliano, M.A., Juliano, L., Oliveira, V., and Machado, M.F.M. (2017). Processing of metacaspase 2 from Trypanosoma brucei (TbMCA2) broadens its substrate specificity. Bba-Proteins Proteom 1865:388-394. Gilroy, S., Suzuki, N., Miller, G., Choi, W.G., Toyota, M., Devireddy, A.R., and Mittler, R. (2014). A tidal wave of signals: calcium and ROS at the forefront of rapid systemic signaling. Trends in Plant Science 19:623-630. Gomez-Gomez, L., and Boller, T. (2000). FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5:1003-1011. Gou, X.P., Yin, H.J., He, K., Du, J.B., Yi, J., Xu, S.B., Lin, H.H., Clouse, S.D., and Li, J. (2012). Genetic Evidence for an Indispensable Role of Somatic Embryogenesis Receptor Kinases in Brassinosteroid Signaling. Plos Genetics 8. Gudynaite-Savitch, L., Johnson, D.A., and Miki, B.L.A. (2009). Strategies to mitigate transgene-promoter interactions. Plant Biotechnol J 7:472-485. Gupta, S., and Dikshit, A. (2009). Biopesticides: An ecofriendly approach for pest control. 3. Hamuel, J.D. (2015). An Overview of Plant Immunity. Journal of Plant Pathology & Microbiology 6. Hander, T., Fernandez-Fernandez, A.D., Kumpf, R.P., Willems, P., Schatowitz, H., Rombaut, D., Staes, A., Nolf, J., Pottie, R., Yao, P., et al. (2019). Damage on plants activates Ca(2+)-dependent metacaspases for release of immunomodulatory peptides. Science 363. He, K., Gou, X., Powell, R.A., Yang, H., Yuan, T., Guo, Z., and Li, J. (2008). Receptor-like protein kinases, BAK1 and BKK1, regulate a light-dependent cell-death control pathway. Plant Signal Behav 3:813-815. 114 He, K., Gou, X., Yuan, T., Lin, H., Asami, T., Yoshida, S., Russell, S.D., and Li, J. (2007). BAK1 and BKK1 regulate brassinosteroid-dependent growth and brassinosteroid-independent cell-death pathways. Curr Biol 17:1109-1115. He, Y.X., Zhou, J.G., Shan, L.B., and Meng, X.Z. (2018). Plant cell surface receptor-mediated signaling - a common theme amid diversity. J Cell Sci 131. Hu, C., Zhu, Y.F., Cui, Y.W., Cheng, K.L., Liang, W., Wei, Z.Y., Zhu, M.S., Yin, H.J., Zeng, L., Xiao, Y., et al. (2018). A group of receptor kinases are essential for CLAVATA signalling to maintain stem cell homeostasis. Nature Plants 4:205-211. James, G.V., Patel, V., Nordstrom, K.J.V., Klasen, J.R., Salome, P.A., Weigel, D., and Schneeberger, K. (2013). User guide for mapping-by-sequencing in Arabidopsis. Genome Biology 14. Jia, Y., McAdams, S.A., Bryan, G.T., Hershey, H.P., and Valent, B. (2000). Direct interaction of resistance gene and avirulence gene products confers rice blast resistance. Embo J 19:4004-4014. Jones, D.A., Thomas, C.M., Hammondkosack, K.E., Balintkurti, P.J., and Jones, J.D.G. (1994). Isolation of the Tomato Cf-9 Gene for Resistance to Cladosporium-Fulvum by Transposon Tagging. Science 266:789-793. Jones, J.D., and Dangl, J.L. (2006). The plant immune system. Nature 444:323-329. Joo, J.H., Wang, S.Y., Chen, J.G., Jones, A.M., and Fedoroff, N.V. (2005). Different signaling and cell death roles of heterotrimeric G protein alpha and beta subunits in the arabidopsis oxidative stress response to ozone. Plant Cell 17:957-970. Kadota, Y., Shirasu, K., and Zipfel, C. (2015). Regulation of the NADPH Oxidase RBOHD During Plant Immunity. Plant Cell Physiol 56:1472-1480. Kadota, Y., Sklenar, J., Derbyshire, P., Stransfeld, L., Asai, S., Ntoukakis, V., Jones, J.D., Shirasu, K., Menke, F., Jones, A., et al. (2014). Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol Cell 54:43-55. Kaminaka, H., Nake, C., Epple, P., Dittgen, J., Schutze, K., Chaban, C., Holt, B.F., 3rd, Merkle, T., Schafer, E., Harter, K., et al. (2006). bZIP10-LSD1 antagonism modulates basal defense and cell death in Arabidopsis following infection. Embo J 25:4400-4411. Karlova, R., Boeren, S., van Dongen, W., Kwaaitaal, M., Aker, J., Vervoort, J., and de Vries, S. (2009). Identification of in vitro phosphorylation sites in the Arabidopsis thaliana somatic embryogenesis receptor-like kinases. Proteomics 9:368-379. Kemmerling, B., Schwedt, A., Rodriguez, P., Mazzotta, S., Frank, M., Abu Qamar, S., Mengiste, T., Betsuyaku, S., Parker, J.E., Mussig, C., et al. (2007). The BRI1-associated kinase 1, BAK1, has a Brassinoli-independent role in plant cell-death control. Current Biology 17:1116-1122. Khan, M.A.S., Chock, P.B., and Stadtman, E.R. (2005). Knockout of caspase-like gene, YCA1, abrogates apoptosis and elevates oxidized proteins in Saccharomyces cerevisiae. P Natl Acad Sci USA 102:17326-17331. Klemencic, M., and Funk, C. (2018a). Structural and functional diversity of caspase homologues in non-metazoan organisms. Protoplasma 255:387-397. Klemencic, M., and Funk, C. (2018b). Type III metacaspases: calcium-dependent activity proposes new function for the p10 domain. New Phytol 218:1179-1191. Klemencic, M., and Funk, C. (2019). Evolution and structural diversity of metacaspases. J Exp Bot. 115 Krasileva, K.V., Dahlbeck, D., and Staskawicz, B.J. (2010). Activation of an Arabidopsis Resistance Protein Is Specified by the in Planta Association of Its Leucine-Rich Repeat Domain with the Cognate Oomycete Effector. Plant Cell 22:2444-2458. Lam, E., and Zhang, Y. (2012). Regulating the reapers: activating metacaspases for programmed cell death. Trends in Plant Science 17:487-494. LaMontagne, E., Collins, C., Peck, S., and Heese, A. (2016). Isolation of Microsomal Membrane Proteins from Arabidopsis thaliana. 217-234. Lee, R.E.C., Brunette, S., Puente, L.G., and Megeney, L.A. (2010). Metacaspase Yca1 is required for clearance of insoluble protein aggregates. P Natl Acad Sci USA 107:13348-13353. Lee, R.E.C., Puente, L.G., Kaern, M., and Megeney, L.A. (2008). A Non-Death Role of the Yeast Metacaspase: Yca1p Alters Cell Cycle Dynamics. Plos One 3. Lehmann, S., Serrano, M., L'Haridon, F., Tjamos, S.E., and Metraux, J.P. (2015). Reactive oxygen species and plant resistance to fungal pathogens. Phytochemistry 112:54-62. Li, B., Ferreira, M.A., Huang, M., Camargos, L.F., Yu, X., Teixeira, R.M., Carpinetti, P.A., Mendes, G.C., Gouveia-Mageste, B.C., Liu, C., et al. (2019). The receptor-like kinase NIK1 targets FLS2/BAK1 immune complex and inversely modulates antiviral and antibacterial immunity. Nat Commun 10:4996. Li, J., Wen, J., Lease, K.A., Doke, J.T., Tax, F.E., and Walker, J.C. (2002a). BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell 110:213-222. Li, J., Wen, J.Q., Lease, K.A., Doke, J.T., Tax, F.E., and Walker, J.C. (2002b). BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell 110:213-222. Li, J.M., and Chory, J. (1997). A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90:929-938. Li, L., Li, M., Yu, L.P., Zhou, Z.Y., Liang, X.X., Liu, Z.X., Cai, G.H., Gao, L.Y., Zhang, X.J., Wang, Y.C., et al. (2014). The FLS2-Associated Kinase BIK1 Directly Phosphorylates the NADPH Oxidase RbohD to Control Plant Immunity. Cell Host Microbe 15:329-338. Li, L., Yu, Y., Zhou, Z., and Zhou, J.M. (2016). Plant pattern-recognition receptors controlling innate immunity. Sci China Life Sci 59:878-888. Li, X., and Zhang, Y. (2016). Suppressor Screens in Arabidopsis. Methods Mol Biol 1363:1-8. Liang, X., Ding, P., Lian, K., Wang, J., Ma, M., Li, L., Li, L., Li, M., Zhang, X., Chen, S., et al. (2016). Arabidopsis heterotrimeric G proteins regulate immunity by directly coupling to the FLS2 receptor. Elife 5:e13568. Liebrand, T.W., van den Berg, G.C., Zhang, Z., Smit, P., Cordewener, J.H., America, A.H., Sklenar, J., Jones, A.M., Tameling, W.I., Robatzek, S., et al. (2013). Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection. Proc Natl Acad Sci U S A 110:10010-10015. Liebrand, T.W., van den Burg, H.A., and Joosten, M.H. (2014a). Two for all: receptor-associated kinases SOBIR1 and BAK1. Trends Plant Sci 19:123-132. Liebrand, T.W.H., van den Burg, H.A., and Joosten, M.H.A.J. (2014b). Two for all: receptor-associated kinases SOBIR1 and BAK1. Trends in Plant Science 19:123-132. 116 Liu, J., Ding, P., Sun, T., Nitta, Y., Dong, O., Huang, X., Yang, W., Li, X., Botella, J.R., and Zhang, Y. (2013a). Heterotrimeric G proteins serve as a converging point in plant defense signaling activated by multiple receptor-like kinases. Plant Physiol 161:2146-2158. Liu, J., Elmore, J.M., Lin, Z.J.D., and Coaker, G. (2011). A Receptor-like Cytoplasmic Kinase Phosphorylates the Host Target RIN4, Leading to the Activation of a Plant Innate Immune Receptor. Cell Host Microbe 9:137-146. Liu, J.M., Ding, P.T., Sun, T.J., Nitta, Y., Dong, O., Huang, X.C., Yang, W., Li, X., Botella, J.R., and Zhang, Y.L. (2013b). Heterotrimeric G Proteins Serve as a Converging Point in Plant Defense Signaling Activated by Multiple Receptor-Like Kinases. Plant Physiology 161:2146-2158. Liu, T.L., Song, T.Q., Zhang, X., Yuan, H.B., Su, L.M., Li, W.L., Xu, J., Liu, S.H., Chen, L.L., Chen, T.Z., et al. (2014). Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis. Nature Communications 5. Liu, Y., Huang, X., Li, M., He, P., and Zhang, Y. (2016). Loss-of-function of Arabidopsis receptor-like kinase BIR1 activates cell death and defense responses mediated by BAK1 and SOBIR1. New Phytol 212:637-645. Liu, Y., Ren, D., Pike, S., Pallardy, S., Gassmann, W., and Zhang, S. (2007). Chloroplast-generated reactive oxygen species are involved in hypersensitive response-like cell death mediated by a mitogen-activated protein kinase cascade. Plant J 51:941-954. Lu, D., Wu, S., Gao, X., Zhang, Y., Shan, L., and He, P. (2010a). A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proc Natl Acad Sci U S A 107:496-501. Lu, D.P., Wu, S.J., Gao, X.Q., Zhang, Y.L., Shan, L.B., and He, P. (2010b). A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. P Natl Acad Sci USA 107:496-501. Ma, X., Xu, G., He, P., and Shan, L. (2016a). SERKing Coreceptors for Receptors. Trends Plant Sci 21:1017-1033. Ma, X.Y., Xu, G.Y., He, P., and Shan, L.B. (2016b). SERKing Coreceptors for Receptors. Trends in Plant Science 21:1017-1033. Machado, M.F., Marcondes, M.F., Juliano, M.A., McLuskey, K., Mottram, J.C., Moss, C.X., Juliano, L., and Oliveira, V. (2013). Substrate specificity and the effect of calcium on Trypanosoma brucei metacaspase 2. FEBS J 280:2608-2621. Macho, A.P., and Zipfel, C. (2014). Plant PRRs and the activation of innate immune signaling. Mol Cell 54:263-272. MacKenzie, S.H., and Clark, A.C. (2012). Death by Caspase Dimerization. Adv Exp Med Biol 747:55-73. Mackey, D., Holt, B.F., Wiig, A., and Dangl, J.L. (2002). RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis. Cell 108:743-754. Mazzoni, C., and Falcone, C. (2008). Caspase-dependent apoptosis in yeast. Bba-Mol Cell Res 1783:1320-1327. McKenna, A., Hanna, M., Banks, E., Sivachenko, A., Cibulskis, K., Kernytsky, A., Garimella, K., Altshuler, D., Gabriel, S., Daly, M., et al. (2010). The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297-1303. 117 McLuskey, K., Rudolf, J., Proto, W.R., Isaacs, N.W., Coombs, G.H., Moss, C.X., and Mottram, J.C. (2012). Crystal structure of a Trypanosoma brucei metacaspase. Proc Natl Acad Sci U S A 109:7469-7474. Meng, X.Z., Chen, X., Mang, H.G., Liu, C.L., Yu, X., Gao, X.Q., Torii, K.U., He, P., and Shan, L.B. (2015). Differential Function of Arabidopsis SERK Family Receptor-like Kinases in Stomatal Patterning. Current Biology 25:2361-2372. Meng, X.Z., and Zhang, S.Q. (2013). MAPK Cascades in Plant Disease Resistance Signaling. Annu Rev Phytopathol 51:245-266. Monaghan, J., Matschi, S., Shorinola, O., Rovenich, H., Matei, A., Segonzac, C., Malinovsky, F.G., Rathjen, J.P., MacLean, D., Romeis, T., et al. (2014). The Calcium-Dependent Protein Kinase CPK28 Buffers Plant Immunity and Regulates BIK1 Turnover. Cell Host Microbe 16:605-615. Moss, C.X., Westrop, G.D., Juliano, L., Coombs, G.H., and Mottram, J.C. (2007). Metacaspase 2 of Trypanosoma brucei is a calcium-dependent cysteine peptidase active without processing. Febs Lett 581:5635-5639. Mukhtar, M.S., Carvunis, A.R., Dreze, M., Epple, P., Steinbrenner, J., Moore, J., Tasan, M., Galli, M., Hao, T., Nishimura, M.T., et al. (2011). Independently Evolved Virulence Effectors Converge onto Hubs in a Plant Immune System Network. Science 333:596-601. Nagata, S. (2018). Apoptosis and Clearance of Apoptotic Cells. Annu Rev Immunol 36:489-517. Nam, K.H., and Li, J. (2002). BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell 110:203-212. Ntoukakis, V., Schwessinger, B., Segonzac, C., and Zipfel, C. (2011). Cautionary notes on the use of C-terminal BAK1 fusion proteins for functional studies. Plant Cell 23:3871-3878. Oh, E.S., Lee, Y., Chae, W.B., Rameneni, J.J., Park, Y.S., Lim, Y.P., and Oh, M.H. (2018). Biochemical Analysis of the Role of Leucine-Rich Repeat Receptor-Like Kinases and the Carboxy-Terminus of Receptor Kinases in Regulating Kinase Activity in Arabidopsis thaliana and Brassica oleracea. Molecules 23. Opitz, R., Muller, M., Reuter, C., Barone, M., Soicke, A., Roske, Y., Piotukh, K., Huy, P., Beerbaum, M., Wiesner, B., et al. (2015). A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions. Proc Natl Acad Sci U S A 112:5011-5016. Peng, Y., van Wersch, R., and Zhang, Y. (2018). Convergent and Divergent Signaling in PAMP-Triggered Immunity and Effector-Triggered Immunity. Mol Plant Microbe Interact 31:403-409. Perraki, A., DeFalco, T.A., Derbyshire, P., Avila, J., Sere, D., Sklenar, J., Qi, X.Y., Stransfeld, L., Schwessinger, B., Kadota, Y., et al. (2018). Phosphocode-dependent functional dichotomy of a common co-receptor in plant signalling. Nature 561:248-+. Qi, J.S., Wang, J.L., Gong, Z.Z., and Zhou, J.M. (2017). Apoplastic ROS signaling in plant immunity. Curr Opin Plant Biol 38:92-100. Qiu, J.L., Fiil, B.K., Petersen, K., Nielsen, H.B., Botanga, C.J., Thorgrimsen, S., Palma, K., Suarez-Rodriguez, M.C., Sandbech-Clausen, S., Lichota, J., et al. (2008). Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus. Embo J 27:2214-2221. Ranf, S., Eschen-Lippold, L., Pecher, P., Lee, J., and Scheel, D. (2011). Interplay between calcium signalling and early signalling elements during defence responses to microbe- or damage-associated molecular patterns. Plant Journal 68:100-113. 118 Rawlings, N.D., Alan, J., Thomas, P.D., Huang, X.D., Bateman, A., and Finn, R.D. (2018). The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res 46:D624-D632. Rawlings, N.D., Barrett, A.J., and Bateman, A. (2010). MEROPS: the peptidase database. Nucleic Acids Res 38:D227-233. Rekhter, D., Ludke, D., Ding, Y., Feussner, K., Zienkiewicz, K., Lipka, V., Wiermer, M., Zhang, Y., and Feussner, I. (2019). Isochorismate-derived biosynthesis of the plant stress hormone salicylic acid. Science 365:498-502. Rivas, S., and Thomas, C.M. (2005). Molecular interactions between tomato and the leaf mold pathogen Cladosporium fulvum. Annual Review of Phytopathology 43:395-436. Rodriguez, M.C., Petersen, M., and Mundy, J. (2010a). Mitogen-activated protein kinase signaling in plants. Annu Rev Plant Biol 61:621-649. Rodriguez, M.C.S., Petersen, M., and Mundy, J. (2010b). Mitogen-Activated Protein Kinase Signaling in Plants. Annu Rev Plant Biol 61:621-649. Roux, M., Schwessinger, B., Albrecht, C., Chinchilla, D., Jones, A., Holton, N., Malinovsky, F.G., Tor, M., de Vries, S., and Zipfel, C. (2011a). The Arabidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens. Plant Cell 23:2440-2455. Roux, M., Schwessinger, B., Albrecht, C., Chinchilla, D., Jones, A., Holton, N., Malinovsky, F.G., Tor, M., de Vries, S., and Zipfel, C. (2011b). The Arabidopsis Leucine-Rich Repeat Receptor-Like Kinases BAK1/SERK3 and BKK1/SERK4 Are Required for Innate Immunity to Hemibiotrophic and Biotrophic Pathogens. Plant Cell 23:2440-2455. Salvesen, G.S., Hempel, A., and Coll, N.S. (2016a). Protease signaling in animal and plant-regulated cell death. FEBS J 283:2577-2598. Salvesen, G.S., Hempel, A., and Coll, N.S. (2016b). Protease signaling in animal and plant-regulated cell death. Febs Journal 283:2577-2598. Sarris, P.F., Duxbury, Z., Huh, S.U., Ma, Y., Segonzac, C., Sklenar, J., Derbyshire, P., Cevik, V., Rallapalli, G., Saucet, S.B., et al. (2015). A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors. Cell 161:1089-1100. Savary, S., Ficke, A., Aubertot, J.N., and Hollier, C. (2012). Crop losses due to diseases and their implications for global food production losses and food security. Food Secur 4:519-537. Schlessinger, J. (2000). Cell signaling by receptor tyrosine kinases. Cell 103:211-225. Schulze, B., Mentzel, T., Jehle, A.K., Mueller, K., Beeler, S., Boller, T., Felix, G., and Chinchilla, D. (2010). Rapid Heteromerization and Phosphorylation of Ligand-activated Plant Transmembrane Receptors and Their Associated Kinase BAK1. Journal of Biological Chemistry 285:9444-9451. Schwessinger, B., and Rathjen, J.P. (2015). Changing SERKs and priorities during plant life. Trends in Plant Science 20:531-533. Schwessinger, B., Roux, M., Kadota, Y., Ntoukakis, V., Sklenar, J., Jones, A., and Zipfel, C. (2011). Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1. PLoS Genet 7:e1002046. Segonzac, C., Macho, A.P., Sanmartin, M., Ntoukakis, V., Sanchez-Serrano, J.J., and Zipfel, C. (2014). Negative control of BAK1 by protein phosphatase 2A during plant innate immunity. Embo J 33:2069-2079. 119 Seybold, H., Trempel, F., Ranf, S., Scheel, D., Romeis, T., and Lee, J. (2014). Ca2+ signalling in plant immune response: from pattern recognition receptors to Ca2+ decoding mechanisms. New Phytologist 204:782-790. Shen, W., Liu, J., and Li, J.F. (2019). Type-II Metacaspases Mediate the Processing of Plant Elicitor Peptides in Arabidopsis. Mol Plant. Shi, Y.G. (2004). Caspase activation, inhibition, and reactivation: A mechanistic view. Protein Sci 13:1979-1987. Shiu, S.H., and Bleecker, A.B. (2001). Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proc Natl Acad Sci U S A 98:10763-10768. Shiu, S.H., Karlowski, W.M., Pan, R.S., Tzeng, Y.H., Mayer, K.F.X., and Li, W.H. (2004). Comparative analysis of the receptor-like kinase family in Arabidopsis and rice. Plant Cell 16:1220-1234. Shpak, E.D., McAbee, J.M., Pillitteri, L.J., and Torii, K.U. (2005). Stomatal patterning and differentiation by synergistic interactions of receptor kinases. Science 309:290-293. Shrestha, A., Brunette, S., Stanford, W.L., and Megeney, L.A. (2019). The metacaspase Yca1 maintains proteostasis through multiple interactions with the ubiquitin system. Cell Discov 5. Smotrys, J.E., and Linder, M.E. (2004). Palmitoylation of intracellular signaling proteins: Regulation and function. Annu Rev Biochem 73:559-587. Su, J.B., Yang, L.Y., Zhu, Q.K., Wu, H.J., He, Y., Liu, Y.D., Xu, J., Jiang, D.A., and Zhang, S.Q. (2018). Active photosynthetic inhibition mediated by MPK3/MPK6 is critical to effector-triggered immunity. Plos Biol 16. Sueldo, D.J., and van der Hoorn, R.A.L. (2017). Plant life needs cell death, but does plant cell death need Cys proteases? FEBS J 284:1577-1585. Sun, T., Zhang, Y., Li, Y., Zhang, Q., Ding, Y., and Zhang, Y. (2015). ChIP-seq reveals broad roles of SARD1 and CBP60g in regulating plant immunity. Nat Commun 6:10159. Sun, T.J., Nitta, Y., Zhang, Q., Wu, D., Tian, H.N., Lee, J.S., and Zhang, Y.L. (2018). Antagonistic interactions between two MAP kinase cascades in plant development and immune signaling. Embo Reports 19. Sun, Y.D., Han, Z.F., Tang, J., Hu, Z.H., Chai, C.L., Zhou, B., and Chai, J.J. (2013a). Structure reveals that BAK1 as a co-receptor recognizes the BRI1-bound brassinolide. Cell Res 23:1326-1329. Sun, Y.D., Li, L., Macho, A.P., Han, Z.F., Hu, Z.H., Zipfel, C., Zhou, J.M., and Chai, J.J. (2013b). Structural Basis for flg22-Induced Activation of the Arabidopsis FLS2-BAK1 Immune Complex. Science 342:624-628. ThordalChristensen, H., Zhang, Z.G., Wei, Y.D., and Collinge, D.B. (1997). Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant Journal 11:1187-1194. Tian, W., Hou, C., Ren, Z., Wang, C., Zhao, F., Dahlbeck, D., Hu, S., Zhang, L., Niu, Q., Li, L., et al. (2019). A calmodulin-gated calcium channel links pathogen patterns to plant immunity. Nature 572:131-135. Trinkle-Mulcahy, L. (2019). Recent advances in proximity-based labeling methods for interactome mapping. F1000Res 8. 120 Tsiatsiani, L., Timmerman, E., De Bock, P.J., Vercammen, D., Stael, S., van de Cotte, B., Staes, A., Goethals, M., Beunens, T., Van Damme, P., et al. (2013). The Arabidopsis metacaspase9 degradome. Plant Cell 25:2831-2847. Tsiatsiani, L., Van Breusegem, F., Gallois, P., Zavialov, A., Lam, E., and Bozhkov, P.V. (2011). Metacaspases. Cell Death Differ 18:1279-1288. Tsuda, K., Mine, A., Bethke, G., Igarashi, D., Botanga, C.J., Tsuda, Y., Glazebrook, J., Sato, M., and Katagiri, F. (2013). Dual Regulation of Gene Expression Mediated by Extended MAPK Activation and Salicylic Acid Contributes to Robust Innate Immunity in Arabidopsis thaliana. Plos Genetics 9. Uren, A.G., O'Rourke, K., Aravind, L.A., Pisabarro, M.T., Seshagiri, S., Koonin, E.V., and Dixit, V.M. (2000). Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma. Mol Cell 6:961-967. van der Hoorn, R.A., and Kamoun, S. (2008). From Guard to Decoy: a new model for perception of plant pathogen effectors. Plant Cell 20:2009-2017. van der Hoorn, R.A.L. (2008). Plant proteases: From phenotypes to molecular mechanisms. Annual Review of Plant Biology 59:191-223. van Wersch, R., Li, X., and Zhang, Y.L. (2016). Mighty Dwarfs: Arabidopsis Autoimmune Mutants and Their Usages in Genetic Dissection of Plant Immunity. Frontiers in Plant Science 7. Veitia, R.A. (2007). Exploring the molecular etiology of dominant-negative mutations. Plant Cell 19:3843-3851. Vercammen, D., van de Cotte, B., De Jaeger, G., Eeckhout, D., Casteels, P., Vandepoele, K., Vandenberghe, I., Van Beeumen, J., Inze, D., and Van Breusegem, F. (2004). Type II metacaspases Atmc4 and Atmc9 of Arabidopsis thaliana cleave substrates after arginine and lysine. J Biol Chem 279:45329-45336. Wang, J.Z., Li, H.J., Han, Z.F., Zhang, H.Q., Wang, T., Lin, G.Z., Chang, J.B., Yang, W.C., and Chai, J.J. (2015). Allosteric receptor activation by the plant peptide hormone phytosulfokine. Nature 525:265-+. Wang, S., Narendra, S., and Fedoroff, N. (2007). Heterotrimeric G protein signaling in the Arabidopsis unfolded protein response. Proc Natl Acad Sci U S A 104:3817-3822. Wang, X., Li, X., Meisenhelder, J., Hunter, T., Yoshida, S., Asami, T., and Chory, J. (2005). Autoregulation and homodimerization are involved in the activation of the plant steroid receptor BRI1. Dev Cell 8:855-865. Wang, X., Wang, X., Feng, H., Tang, C., Bai, P., Wei, G., Huang, L., and Kang, Z. (2012). TaMCA4, a novel wheat metacaspase gene functions in programmed cell death induced by the fungal pathogen Puccinia striiformis f. sp. tritici. Mol Plant Microbe Interact 25:755-764. Watanabe, N., and Lam, E. (2011a). Arabidopsis metacaspase 2d is a positive mediator of cell death induced during biotic and abiotic stresses. Plant Journal 66:969-982. Watanabe, N., and Lam, E. (2011b). Calcium-dependent activation and autolysis of Arabidopsis metacaspase 2d. J Biol Chem 286:10027-10040. Weigel, D., Ahn, J.H., Blazquez, M.A., Borevitz, J.O., Christensen, S.K., Fankhauser, C., Ferrandiz, C., Kardailsky, I., Malancharuvil, E.J., Neff, M.M., et al. (2000). Activation tagging in Arabidopsis. Plant Physiology 122:1003-1013. 121 Wessling, R., Epple, P., Altmann, S., He, Y.J., Yang, L., Henz, S.R., McDonald, N., Wiley, K., Bader, K.C., Glasser, C., et al. (2014). Convergent Targeting of a Common Host Protein-Network by Pathogen Effectors from Three Kingdoms of Life. Cell Host Microbe 16:364-375. Wiermer, M., Feys, B.J., and Parker, J.E. (2005). Plant immunity: the EDS1 regulatory node. Curr Opin Plant Biol 8:383-389. Williamson, M.P. (1994). The Structure and Function of Proline-Rich Regions in Proteins. Biochem J 297:249-260. Winter, D., Vinegar, B., Nahal, H., Ammar, R., Wilson, G.V., and Provart, N.J. (2007). An \"Electronic Fluorescent Pictograph\" Browser for Exploring and Analyzing Large-Scale Biological Data Sets. Plos One 2. Wong, A.H., Yan, C., and Shi, Y. (2012). Crystal structure of the yeast metacaspase Yca1. J Biol Chem 287:29251-29259. Wrzaczek, M., Vainonen, J.P., Stael, S., Tsiatsiani, L., Help-Rinta-Rahko, H., Gauthier, A., Kaufholdt, D., Bollhoner, B., Lamminmaki, A., Staes, A., et al. (2015). GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis. Embo J 34:55-66. Wu, D., Liu, Y., Xu, F., and Zhang, Y. (2018). Differential requirement of BAK1 C-terminal tail in development and immunity. J Integr Plant Biol 60:270-275. Wu, F.H., Shen, S.C., Lee, L.Y., Lee, S.H., Chan, M.T., and Lin, C.S. (2009). Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method. Plant Methods 5:16. Wu, W.Z., Wu, Y.J., Gao, Y., Li, M.Z., Yin, H.J., Lv, M.H., Zhao, J.X., Li, J., and He, K. (2015). Somatic embryogenesis receptor-like kinase 5 in the ecotype Landsberg erecta of Arabidopsis is a functional RD LRR-RLK in regulating brassinosteroid signaling and cell death control. Frontiers in Plant Science 6. Wu, Y., Zhang, D., Chu, J.Y., Boyle, P., Wang, Y., Brindle, I.D., De Luca, V., and Despres, C. (2012). The Arabidopsis NPR1 Protein Is a Receptor for the Plant Defense Hormone Salicylic Acid. Cell Reports 1:639-647. Xin, X.F., Nomura, K., Aung, K., Velasquez, A.C., Yao, J., Boutrot, F., Chang, J.H., Zipfel, C., and He, S.Y. (2016). Bacteria establish an aqueous living space in plants crucial for virulence. Nature 539:524-+. Xing, H.L., Dong, L., Wang, Z.P., Zhang, H.Y., Han, C.Y., Liu, B., Wang, X.C., and Chen, Q.J. (2014). A CRISPR/Cas9 toolkit for multiplex genome editing in plants. Bmc Plant Biology 14. Xu, F., Copeland, C., and Li, X. (2015). Protein Immunoprecipitation Using Nicotiana benthamiana Transient Expression System. BIO-PROTOCOL 5. Xu, F. (2017). Functional analysis of a plant metacaspase in negative regulation of innate immunity. Electronic Theses and Dissertation (ETDs) 2008+. University of British Columbia. Xu, T.D., Dai, N., Chen, J.S., Nagawa, S., Cao, M., Li, H.J., Zhou, Z.M., Chen, X., De Rycke, R., Rakusova, H., et al. (2014). Cell Surface ABP1-TMK Auxin-Sensing Complex Activates ROP GTPase Signaling. Science 343:1025-1028. Yamaguchi, Y., Huffaker, A., Bryan, A.C., Tax, F.E., and Ryan, C.A. (2010). PEPR2 Is a Second Receptor for the Pep1 and Pep2 Peptides and Contributes to Defense Responses in Arabidopsis. Plant Cell 22:508-522. 122 Yamaguchi, Y., Pearce, G., and Ryan, C.A. (2006). The cell surface leucine-rich repeat receptor for AtPep1, an endogenous peptide elicitor in Arabidopsis, is functional in transgenic tobacco cells. Proc Natl Acad Sci U S A 103:10104-10109. Yang, B., Sugio, A., and White, F.F. (2006). Os8N3 is a host disease-susceptibility gene for bacterial blight of rice. P Natl Acad Sci USA 103:10503-10508. Yang, B., Zhu, W.G., Johnson, L.B., and White, F.F. (2000). The virulence factor AvrXa7 of Xanthomonas oryzae pv, oryzae is a type III secretion pathway-dependent nuclear-localized double-stranded DNA-binding protein. P Natl Acad Sci USA 97:9807-9812. Yoo, S.Y., Bomblies, K., Yoo, S.K., Yang, J.W., Choi, M.S., Lee, J.S., Weigel, D., and Ahn, J.H. (2005). The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta 221:523-530. Yu, X., Xu, G., Li, B., de Souza Vespoli, L., Liu, H., Moeder, W., Chen, S., de Oliveira, M.V.V., Ariadina de Souza, S., Shao, W., et al. (2019). The Receptor Kinases BAK1/SERK4 Regulate Ca(2+) Channel-Mediated Cellular Homeostasis for Cell Death Containment. Curr Biol 29:3778-3790 e3778. Zhang, S.Q., and Klessig, D.F. (1998). Resistance gene N-mediated de novo synthesis and activation of a tobacco mitogen-activated protein kinase by tobacco mosaic virus infection. P Natl Acad Sci USA 95:7433-7438. Zhang, W.G., Fraiture, M., Kolb, D., Loffelhardt, B., Desaki, Y., Boutrot, F.F.G., Tor, M., Zipfel, C., Gust, A.A., and Brunner, F. (2013). Arabidopsis RECEPTOR-LIKE PROTEIN30 and Receptor-Like Kinase SUPPRESSOR OF BIR1-1/EVERSHED Mediate Innate Immunity to Necrotrophic Fungi. Plant Cell 25:4227-4241. Zhang, Y., and Li, X. (2019). Salicylic acid: biosynthesis, perception, and contributions to plant immunity. Curr Opin Plant Biol 50:29-36. Zhang, Y.X., Xu, S.H., Ding, P.T., Wang, D.M., Cheng, Y.T., He, J., Gao, M.H., Xu, F., Li, Y., Zhu, Z.H., et al. (2010). Control of salicylic acid synthesis and systemic acquired resistance by two members of a plant-specific family of transcription factors. P Natl Acad Sci USA 107:18220-18225. Zhang, Z., Liu, Y., Huang, H., Gao, M., Wu, D., Kong, Q., and Zhang, Y. (2017). The NLR protein SUMM2 senses the disruption of an immune signaling MAP kinase cascade via CRCK3. EMBO Rep 18:292-302. Zhang, Z., Wu, Y., Gao, M., Zhang, J., Kong, Q., Liu, Y., Ba, H., Zhou, J., and Zhang, Y. (2012). Disruption of PAMP-induced MAP kinase cascade by a Pseudomonas syringae effector activates plant immunity mediated by the NB-LRR protein SUMM2. Cell Host Microbe 11:253-263. Zhao, D.Z., Wang, G.F., Speal, B., and Ma, H. (2002). The EXCESS MICROSPOROCYTES1 gene encodes a putative leucine-rich repeat receptor protein kinase that controls somatic and reproductive cell fates in the Arabidopsis anther. Gene Dev 16:2021-2031. Zhu, J.Y., Sae-Seaw, J., and Wang, Z.Y. (2013). Brassinosteroid signalling. Development 140:1615-1620. Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J.D., Boller, T., and Felix, G. (2006a). Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125:749-760. 123 Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J.D.G., Boller, T., and Felix, G. (2006b). Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125:749-760. "@en . "Thesis/Dissertation"@en . "2020-05"@en . "10.14288/1.0388218"@en . "eng"@en . "Botany"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "Attribution-NonCommercial-NoDerivatives 4.0 International"@* . "http://creativecommons.org/licenses/by-nc-nd/4.0/"@* . "Graduate"@en . "Regulation of the pattern-recognition receptor signaling in Arabidopsis : lessons from sobir7-1 and MC2"@en . "Text"@en . "http://hdl.handle.net/2429/73244"@en .