Science, Faculty of
Botany, Department of
DSpace
UBCV
Wang, Yiran
Doctor of Philosophy - PhD
University of British Columbia
Plants face constant threats from various microbial pathogens. Successful sensing and defending against infections are essential to their survival. Arabidopsis thaliana receptor like protein SUPPRESSOR OF NPR1-1, CONSTITUTIVE 2 (SNC2) is of great importance to plant basal immunity. The transcription factors CALMODULIN-BINDING PROTEIN 60g (CBP60g) and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) define two parallel signaling pathways downstream of SNC2 to activate defense responses. In my PhD research, I aimed to identify the components that are involved in the CBP60g-dependent signaling pathway downstream of SNC2. From a forward genetic screen, mutants with suppressed autoimmunity of sard1-1 snc2-1D were isolated. Characterization of suppressor mutant 125-1 revealed that Epsin-like clathrin adaptor ECA4 is an inhibiting factor of SNC2-mediated immunity. A gain-of-function allele eca4-4D or overexpression of ECA4 leads to enhanced susceptibility against pathogens. Transient expression of ECA4 or eca4-4D in Nicotiana benthamiana reduces the protein levels of BDA1, a transmembrane protein required for SNC2-mediated immunity. Additionally, eca4-4D partially suppresses the autoimmunity of bda1-17D. Taken together, ECA4 contributes to SNC2-mediated immunity by negatively modulating the protein level of BDA1. 116-1 is a partial suppressor of sard1-1 snc2-1D autoimmunity, which carries a premature stop codon in ZINC-FINGER AND OCRE DOMAIN-CONTAINING PROTEIN 1 (ZOP1). ZOP1 encodes a pre-mRNA splicing factor that is involved in gene regulation. zop1 mutant exhibits enhanced susceptibility against pathogens. Moreover, many defense-related genes, including BDA1, were downregulated in the zop1 plants. Altogether, ZOP1 positively contributes to plant immunity through regulating the transcript levels of defense related genes. Studies of the suppressor mutant 123-1 uncovered the role of deubiquitinating enzyme ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM 1 (AMSH1) in SNC2-mediated immunity. Loss-of-function mutation in AMSH1 caused partial suppression of sard1-1 snc2-1D’s autoimmunity. BDA1 might be a substrate of AMSH1, as silencing AMSH1 homologs in Nicotiana benthamiana results in lower BDA1 protein levels. Thus, AMSH1 likely contributes to immunity regulation through controlling the stability of BDA1. In summary, my Ph.D. research expands the understanding of regulators involved in the CBP60g-dependent signaling pathway downstream of SNC2 and emphasizes the importance of homeostasis control of key immune regulators, such as BDA1.
GENETIC ANALYSIS OF DEFENSE PATHWAY DOWNSTREAM OF THE IMMUNE RECEPTOR SNC2 IN ARABIDOPSIS THALIANA by Yiran Wang B. Sc., China Agricultural University, 2018 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) October 2024 © Yiran Wang, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Genetic analysis of defense pathway downstream of the immune receptor SNC2 in Arabidopsis thaliana submitted by Yiran Wang in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Botany Examining Committee: Dr. Yuelin Zhang, Professor, Botany, UBC Supervisor Dr. George Haughn, Professor Emeritus, Botany, UBC Supervisory Committee Member Dr. Richard Hamelin, Professor, Forest Conservation Sciences, UBC University Examiner Dr. Siyun Wang, Professor, Land and Food Systems, UBC University Examiner Additional Supervisory Committee Members: Dr. Xin Li, Professor, Botany, UBC Co-Supervisor Dr. Liang Song, Assistant Professor, Botany, UBC Supervisory Committee Member iii Abstract Plants face constant threats from various microbial pathogens. Successful sensing and defending against infections are essential to their survival. Arabidopsis thaliana receptor like protein SUPPRESSOR OF NPR1-1, CONSTITUTIVE 2 (SNC2) is of great importance to plant basal immunity. The transcription factors CALMODULIN-BINDING PROTEIN 60g (CBP60g) and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) define two parallel signaling pathways downstream of SNC2 to activate defense responses. In my PhD research, I aimed to identify the components that are involved in the CBP60g-dependent signaling pathway downstream of SNC2. From a forward genetic screen, mutants with suppressed autoimmunity of sard1-1 snc2-1D were isolated. Characterization of suppressor mutant 125-1 revealed that Epsin-like clathrin adaptor ECA4 is an inhibiting factor of SNC2-mediated immunity. A gain-of-function allele eca4-4D or overexpression of ECA4 leads to enhanced susceptibility against pathogens. Transient expression of ECA4 or eca4-4D in Nicotiana benthamiana reduces the protein levels of BDA1, a transmembrane protein required for SNC2-mediated immunity. Additionally, eca4-4D partially suppresses the autoimmunity of bda1-17D. Taken together, ECA4 contributes to SNC2-mediated immunity by negatively modulating the protein level of BDA1. 116-1 is a partial suppressor of sard1-1 snc2-1D autoimmunity, which carries a premature stop codon in ZINC-FINGER AND OCRE DOMAIN-CONTAINING PROTEIN 1 (ZOP1). ZOP1 encodes a pre-mRNA splicing factor that is involved in gene regulation. zop1 mutant exhibits enhanced susceptibility against pathogens. Moreover, many defense-related genes, including iv BDA1, were downregulated in the zop1 plants. Altogether, ZOP1 positively contributes to plant immunity through regulating the transcript levels of defense related genes. Studies of the suppressor mutant 123-1 uncovered the role of deubiquitinating enzyme ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM 1 (AMSH1) in SNC2-mediated immunity. Loss-of-function mutation in AMSH1 caused partial suppression of sard1-1 snc2-1D’s autoimmunity. BDA1 might be a substrate of AMSH1, as silencing AMSH1 homologs in Nicotiana benthamiana results in lower BDA1 protein levels. Thus, AMSH1 likely contributes to immunity regulation through controlling the stability of BDA1. In summary, my Ph.D. research expands the understanding of regulators involved in the CBP60g-dependent signaling pathway downstream of SNC2 and emphasizes the importance of homeostasis control of key immune regulators, such as BDA1. v Lay Summary Plant diseases of major crops cause devastating loss of agricultural productivity. To effectively defend against pathogen infections, plants deploy immune receptors to sense the pathogens and activate downstream defense responses. The molecular mechanisms of immune receptor mediating defense responses are not fully understood. Research in this dissertation identified three immune regulators functioning downstream of immune receptor SNC2. The clathrin adaptor ECA4 and the deubiquitinating enzyme AMSH1 differentially modulate the protein abundance of defense-related protein BDA1. The splicing factor ZOP1 regulates the transcript level of BDA1. These findings provide advanced understanding on how plants regulate defense responses and may inform the sustainable solutions to crop protection. vi Preface The work presented in this dissertation was performed from September 2018 through June 2024. Chapter 1 provides a general introduction of plant immunity. Chapter 2, Chapter 3 and Chapter 4 describe the identification and characterization of three suppressor mutants. Chapter 5 concludes the thesis and propose future research directions. Part of Chapter 1 is modified from a published manuscript as below. Weijie Huang*, Yiran Wang*, Xin Li and Yuelin Zhang. Biosynthesis and Regulation of Salicylic Acid and N-Hydroxypipecolic Acid in Plant Immunity. (2020). Mol. Plant. 13, 31–41, https://doi.org/10.1016/j.molp.2019.12.008 (*These authors contributed equally to this article.) • The candidate and Dr. Weijie Huang drafted the manuscript. Dr. Xin Li and Dr. Yuelin Zhang revised the manuscript. Chapter 2: The clathrin adaptor ECA4 regulates plant immunity through modulating BDA1 levels was based on the following unpublished manuscript: Yiran Wang, Weijie Huang, Xin Li and Yuelin Zhang. • The candidate performed most of the experiments. Dr. Weijie Huang helped with the genetic screen. The candidate wrote the manuscript. Dr. Xin Li and Dr. Yuelin Zhang edited the manuscript. Dr. Yuelin Zhang designed and supervised the study. Chapter 3: The pre-mRNA splicing factor ZOP1 contributes to plant immunity regulation was based on the following unpublished manuscript: Yiran Wang, Weijie Huang, Pingyu Zhang, Hainan Tian, Xin Li and Yuelin Zhang. vii • The candidate performed most of the experiments. Dr. Weijie Huang carried out the genetic screen. Dr. Hainan Tian prepared the samples for RNA-seq. Dr. Pingyu Zhang analyzed the RNA-seq data. The candidate drafted the manuscript. Dr. Xin Li and Dr. Yuelin Zhang edited the manuscript. Dr. Yuelin Zhang designed and supervised the study. Chapter 4: The deubiquitinating enzyme AMSH1 contributes plant immunity through regulating the stability of BDA1 was based on the following unpublished manuscript: Yiran Wang, Weijie Huang, Xin Li and Yuelin Zhang. • The candidate conducted most of the experiments under the supervision of Dr. Yuelin Zhang. Dr. Weijie Huang set up the genetic screen conditions. The candidate wrote the manuscript. Dr. Xin Li and Dr. Yuelin Zhang revised the manuscript. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iii Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables .............................................................................................................................. xiii List of Figures ............................................................................................................................. xiv List of Abbreviations .................................................................................................................. xv Acknowledgements .................................................................................................................... xxi Dedication ................................................................................................................................. xxiii Chapter 1: Introduction ........................................................................................................... 1 1.1 Plant diseases ............................................................................................................. 1 1.2 Overview of the plant immune system .................................................................... 1 1.2.1 Preformed physical and chemical barriers .......................................................... 2 1.2.2 PAMP-triggered immunity ................................................................................. 3 1.2.2.1 PAMPs and Pattern recognition receptors ...................................................... 3 1.2.2.2 Activation of the PRR complexes ................................................................... 4 1.2.2.3 Defense responses downstream of PRR complexes ....................................... 5 1.2.3 Effector-triggered susceptibility ......................................................................... 7 1.2.4 Effector-triggered immunity ............................................................................... 8 1.2.4.1 Nucleotide-binding leucine-rich repeat receptors ........................................... 8 1.2.4.2 Effector perception by plant NLRs ................................................................. 8 1.2.4.3 NLRs-mediated immune responses .............................................................. 10 1.3 PTI and ETI crosstalk ............................................................................................ 11 1.4 Systemic acquired resistance ................................................................................. 12 ix 1.4.1 Signaling molecules in SAR ............................................................................. 13 1.5 Biosynthesis and regulation of SA and NHP ........................................................ 14 1.5.1 SA biosynthesis in plants .................................................................................. 14 1.5.2 Transcriptional regulation of SA biosynthetic genes ........................................ 16 1.5.3 SA perception and downstream signaling ......................................................... 18 1.5.4 NHP biosynthesis .............................................................................................. 19 1.5.5 Transcriptional regulation of NHP biosynthetic genes ..................................... 19 1.6 SARD1 and CBP60g ............................................................................................... 20 1.6.1 Regulation of SARD1 and CBP60g .................................................................. 21 1.7 SNC2-mediated immune pathway ......................................................................... 22 1.8 Thesis objectives ...................................................................................................... 25 Chapter 2: The clathrin adaptor ECA4 regulates plant immunity through modulating BDA1 levels .............................................................................................................................. 26 2.1 Summary .................................................................................................................. 26 2.2 Introduction ............................................................................................................. 27 2.3 Material and methods ............................................................................................. 30 2.3.1 Plant materials and growth conditions .............................................................. 30 2.3.2 Generation of deletion mutants ......................................................................... 31 2.3.3 Mutagenesis and suppressor screen .................................................................. 31 2.3.4 Mapping-by-sequencing ................................................................................... 32 2.3.5 Gene expression analysis .................................................................................. 32 2.3.6 Pathogen infection assays ................................................................................. 32 2.3.7 SA extraction and quantification ....................................................................... 33 2.3.8 Transient expression in Nicotiana benthamiana ............................................... 34 2.4 Results ...................................................................................................................... 35 x 2.4.1 Identification and characterization of an EMS suppressor of sard1-1 snc2-1D 35 2.4.2 Mapping-by-sequencing of 125-1 ..................................................................... 36 2.4.3 Deletion mutants of At2g25430 in 125-1 restore the constitutively activated immunity …………………………………………………………………………………38 2.4.4 eca4-4D carries a gain-of-function mutation in ECA4. .................................... 40 2.4.5 Overexpression of ECA4 or eca4-4D reduces BDA1 protein levels ................ 41 2.4.6 eca4-4D partially suppress the autoimmunity of bda1-17D ............................. 43 2.5 Discussion ................................................................................................................ 45 Chapter 3: The pre-mRNA splicing factor ZOP1 contributes to plant immunity regulation ................................................................................................................................. 48 3.1 Summary .................................................................................................................. 48 3.2 Introduction ............................................................................................................. 48 3.3 Materials and Methods ........................................................................................... 51 3.3.1 Plant materials and growth conditions .............................................................. 51 3.3.2 Mutant Screening .............................................................................................. 52 3.3.3 Mapping-by-sequencing ................................................................................... 52 3.3.4 Characterization of mutants .............................................................................. 52 3.3.5 Gene Expression and Splicing Pattern Analysis ............................................... 53 3.3.6 RNA-seq analysis .............................................................................................. 53 3.4 Results ...................................................................................................................... 54 3.4.1 Identification and characterization of suppressor mutant 116-1 ....................... 54 3.4.2 Mapping-by-sequencing of 116-1 ..................................................................... 56 3.4.3 Loss of ZOP1 suppresses the autoimmunity of sard1-1 snc2-1D .................... 57 3.4.4 zop1 single mutants exhibit compromised resistance against pathogen ........... 59 3.4.5 zop1 single mutant has decreased BDA1 transcript level .................................. 60 xi 3.5 Discussion ................................................................................................................ 63 Chapter 4: The deubiquitinating enzyme AMSH1 contributes plant immunity through regulating the stability of BDA1 ............................................................................................ 65 4.1 Summary .................................................................................................................. 65 4.2 Introduction ............................................................................................................. 66 4.3 Material and methods ............................................................................................. 69 4.3.1 Plant materials and growth conditions .............................................................. 69 4.3.2 Mutagenesis and suppressor screen .................................................................. 69 4.3.3 Mapping-by-sequencing ................................................................................... 70 4.3.4 Gene expression analysis .................................................................................. 70 4.3.5 Infection assay .................................................................................................. 70 4.3.6 SA measurement ............................................................................................... 70 4.3.7 RNAi construct design and transient expression in N. benthamiana ................ 71 4.4 Results ...................................................................................................................... 72 4.4.1 Identification and characterization of suppressor mutant 123-1 ....................... 72 4.4.2 Sequencing-by-mapping of 123-1 ..................................................................... 73 4.4.3 Loss of AMSH1 suppresses the autoimmunity of sard1-1 snc2-1D ................ 75 4.4.4 amsh1-1 single mutant exhibits enhanced basal immunity and PTI responses 77 4.4.5 Silencing AMSH1-like genes in N. benthamiana negatively affects BDA1 protein levels ..................................................................................................................... 79 4.5 Discussion ................................................................................................................ 80 Chapter 5: Conclusions and future directions ..................................................................... 83 5.1 Conclusions .............................................................................................................. 83 5.2 Future directions ..................................................................................................... 85 5.2.1 Function of ECA4 in plant immunity ............................................................... 85 xii 5.2.2 Function of ZOP1 in plant immunity ................................................................ 87 5.2.3 Function of AMSH1 in plant immunity ............................................................ 88 5.2.4 Function of BDA1 in plant immunity ............................................................... 88 5.2.5 Identification of signaling partner(s) of SNC2 in plant immunity .................... 89 Bibliography ................................................................................................................................ 91 xiii List of Tables Table 2.1 Primers used in Chapter 2 ............................................................................................. 34 Table 3.1 Primers used in Chapter 3. ............................................................................................ 54 Table 4.1 Primers used in Chapter 4. ............................................................................................ 72 xiv List of Figures Figure 1.1 SNC2-mediated defense signaling. ............................................................................. 24 Figure 2.1 Identification and characterization of the sard1 snc2-1D suppressor mutant 125-1. .. 36 Figure 2.2 Mapping-by-sequencing of mutant 125-1. .................................................................. 37 Figure 2.3 CRISPR deletion alleles of At2g25430 in the 125-1 background restore the autoimmunity of sard1 snc2-1D. .................................................................................................. 39 Figure 2.4 eca4-4D carries a gain-of-function mutation in ECA4. ............................................... 41 Figure 2.5 Overexpression of ECA4 or eca4-4D reduces BDA1 protein levels in N. benthamiana........................................................................................................................................................ 42 Figure 2.6 eca4-4D partially suppress the autoimmunity of bda1-17D. ...................................... 44 Figure 3.1 Identification and characterization of the sard1 snc2-1D suppressor mutant 116-1. .. 55 Figure 3.2 Mapping-by-sequencing of 116-1. .............................................................................. 56 Figure 3.3 Deletion mutants of ZOP1 in sard1-1 snc2-1D suppress its autoimmunity. ............... 58 Figure 3.4 np::ZOP1 complements the defects of the suppressor 116-1. ..................................... 59 Figure 3.5 Compromised immunity in the zop1 mutants. ............................................................. 60 Figure 3.6 Analysis of defense gene expression in the zop1 mutants. .......................................... 62 Figure 4.1 Identification and characterization of the sard1 snc2-1D suppressor mutant 123-1. .. 73 Figure 4.2 Mapping-by-sequencing of 123-1. .............................................................................. 74 Figure 4.3 Deletion mutations of AMSH1 generated through CRISPR suppress the autoimmunity of of sard1-1 snc2-1D. .................................................................................................................. 76 Figure 4.4 pAMSH1::AMSH1 complements the phenotypes of the suppressor 123-1. ................ 77 Figure 4.5 Enhanced basal immunity and PTI in the amsh1-1 single mutant. ............................. 78 Figure 4.6 Silencing AMSH1-like genes in N. benthamiana leads to decreased protein levels of BDA1. ........................................................................................................................................... 80 Figure 5.1 A work model of SNC2-mediated signaling pathway. ................................................ 85 xv List of Abbreviations 35S A strong promoter from cauliflower mosaic virus A. thaliana Arabidopsis thaliana ABA Abscisic acid ABRC Arabidopsis biological resource center ACD6 Accelerated cell death 6 ADP Adenosine diphosphate ADPr-ATP/di-ADPR ADP-ribosylated ATP/ADPR ADR1 Activated disease resistance 1 AIM1 Abnormal inflorescence meristem 1 ALD1 AGD2-like defense response protein 1 AMSH1 Associated molecule with the SH3 domain of STAM 1 ANOVA Analysis of variance ANP2/3 Mitogen-activated protein kinase kinase kinase 2/3 ANTH AP180 N-terminal homology domain AP2 Adaptor protein complex 2 ATP Adenosine triphosphate ATR1 Effector from Hyaloperonospora arabidopsidis AvrPi-Ta Effector from Magnaporthe grisea AvrPphB Effector from Pseudomonas syringae pv. tomato DC3000 AvrPto Effector from Pseudomonas syringae pv. tomato DC3000 AvrPtoB Effector from Pseudomonas syringae pv. tomato DC3000 AvrRpm1 Effector from Pseudomonas syringae pv. tomato DC3000 AvrRps4 Effector from Pseudomonas syringae pv. tomato DC3000 AvrRpt2 Effector from Pseudomonas syringae pv. tomato DC3000 BA Benzoic acid BAK1 BRI1-associated kinase 1 BDA1 Bian Da 1; Bian Da means “becoming big” in Chinese BIK1 Botrytis-induced kinase 1 BOR1 Borate exporter 1 xvi BRI1 Brassinosteroid insensitive 1 BSK1 Brassinosteroid-signaling kinase 1 CA Cinnamic acid CaM Calmodulin CAP1 Clathrin associated protein 1 Cas9 CRISPR-associated 9 CBP60a Calmodulin binding protein 60 a CBP60b Calmodulin binding protein 60 b CBP60g Calmodulin binding protein 60 g CC Coiled-Coil cDNA Complementary DNA CEBiP Chitin elicitor binding protein CERK1 Chitin elicitor receptor kinase 1 ChIP Chromatin immunoprecipitation CME Clathrin-mediated endocytosis CNGC Cyclic nucleotide-gated channel protein CNL CC-NB-LRR protein CPK5 Calmodulin-domain protein kinase 5 CRISPR Clustered regularly interspaced short palindromic repeats CRK Cysteine-rich receptor-like kinase cryo-EM Cryogenic electron microscopy DAVID Database for annotation, visualization and integrated discovery DEGs Differentially expressed genes DUB Deubiquitinating enzyme ECA4 Epsin-like clathrin adaptor 4 EDS1 Enhanced disease susceptibility 1 EDS5 Enhanced disease susceptibility 5 EF-Tu Bacterial elongation factor-Tu EFR EF-Tu receptor elf18 A conserved N-terminal epitope of the bacterial elongation factor Tu EMS Ethyl methane sulfonate xvii ESCRT Endosomal sorting complexes required for transport ETI Effector-triggered immunity ETS Effector-triggered susceptibility FLAG An epitope protein tag composed of DYKDDDDK flg22 A short peptide of the bacterial flagellin protein FLS2 Flagellin-sensitive 2 FMO1 Flavin-dependent monooxygenase 1 GATK Genome analysis toolkit GFP Green fluorescent protein GO Gene ontology GTL1 GT2-like 1 HA An epitope protein tag composed of YPYDVPDYA HISAT2 Hierarchical indexing for spliced alignment of transcripts 2 hNLR Helper NLR HopAI1 Effector from Pseudomonas syringae pv. tomato DC3000 HopF2 Effector from Pseudomonas syringae pv. tomato DC3000 Hpa Hyaloperonospora arabidopsidis HPLC High-performance liquid chromatography HR Hypersensitive response ICS Isochorismate synthase ID Integrated decoy INA 2,6-dichloroisonicotinic acid ITN1 Increased tolerance to NaCl 1 Ler Landsberg erecta LRR Leucine rich repeat LYK5 Lysin motif-containing receptor kinase 5 LysM Lysin motif M. oryzae Magnaporthe oryzae MAPK Mitogen-activated protein kinase MAPKKK Mitogen-activated protein kinase kinase kinase MATE Multidrug and toxic compound extrusion transporter xviii MEKK1 Mitogen-activated protein kinase kinase kinase 1 MKK Mitogen-activated protein kinase kinase MPK Mitogen-activated protein kinase MS Murashige and Skoog N. benthamiana Nicotiana benthamiana NADase NAD+ cleaving enzyme NADPH Reduced nicotinamide adenine dinucleotide phosphate nahG A salicylate hydroxylase identified in bacteria Pseudomonas putida NB Nucleotide-binding NDR1 Non race-specific disease resistance 1 NHP N-hydroxypipecolic acid nlp20 A short peptide of Necrosis and Ethylene-Inducing Peptide 1-Like Protein NLR Nucleotide-binding leucine-rich repeats receptors NPR Non-expressor of pathogenesis-related proteins NRG1 N requirement gene 1 NUDT Nudix hydrolase OD Optical density P. syringae Pseudomonas syringae P2C ∆1-piperideine-2-carboxylic acid PAD4 Phytoalexin deficient 4 PAGE Polyacrylamide gel electrophoresis PAL Phenylalanine ammonia-lyase PAMP Pathogen-associated molecular pattern PBL PBS1-like protein PBS1 AvrPphb susceptible 1 PBS3 AvrPphb susceptible 3 PCR Polymerase chain reaction PCRK PTI compromised receptor-like cytoplasmic kinase 1 PDK Pyruvate orthophosphate dikinase PEPR1 PEP receptor 1 xix Phe Phenylalanine PIN2 PIN-formed 2 Pip L-pipecolic acid PM Plasma membrane PopP2 Effector from Ralstonia solanacearum PR genes Pathogenesis-related genes pRib-AMP/ADP 2’-(5’’-phosphoribosyl)-5’-adenosine monophosphate/diphosphate PRR Pattern recognition receptor Psm ES4326 Pseudomonas syringae pv. maculicola ES4326 Pst DC3000 Pseudomonas syringae pv. tomato DC3000 Pst DC3000 hrcC- Pst DC3000 lacking hrcC gene, defective in Type III secretion system PTI Pathogen-associated molecular pattern-triggered immunity qPCR Quantitative polymerase chain reaction qRT-PCR Quantitative reverse transcript-PCR R proteins Resistance proteins RbohD Respiratory burst oxidase homolog D RdDM RNA-directed DNA methylation RIN4 RPM1 interacting protein 4 RLCK Receptor-like cytoplasmic kinase RLK Receptor like kinase RLP Receptor like protein rMATS Replicate multivariate analysis of transcript splicing RNA Ribonucleic acid RNAi RNA interference ROS Reactive oxygen species RPM1 Resistance to Pseudomonas syringae pv. maculicola 1 RPP1 Recognition of Peronospora Parasitica 1 RPS2 Resistance to Pseudomonas syringae 2 RPS4 Recognition of Peronospora parasitica 4 RRS1 Resistance to Ralstonia solanacearum 1 RT-PCR Reverse transcription PCR xx SA Salicylic acid SAG101 Senescence-associated gene 101 SAR Systemic acquired resistance SARD1 SAR deficient 1 SARD4 SAR deficient 4 SDS Sodium dodecyl sulfate SERK Somatic-embryogenesis receptor-like kinase SID2 SA induction deficient 2 SNC1 Suppressor of npr1-1, constitutive 1 SNC2 Suppressor of npr1-1, constitutive 2 SNP Single nucleotide polymorphism snRNP Small nuclear ribonucleoprotein particles SOBIR1 Suppressor of BAK1 interacting receptor kinase 1 T3SS Type III secretion system TGA TGACG-binding factor TGN/EE Trans-Golgi network/early endosome TIR domain Toll/Interleukin 1 Receptor domain TMV Tobacco mosaic virus TNL TIR-NB-LRR protein TPC TPLATE complex TurboID Biotin ligase Ub Ubiquitin UGT76B1 UDP-dependent glycosyltransferase 76b1 WGS Whole genome sequencing WRKY Domain found in WRKY family of transcription factors ZAR1 HopZ-activated resistance 1 ZOP1 Zinc-finger and Ocre domain-containing Protein1 xxi Acknowledgements With the generous help and support from all the people I met, the past six years of doctoral study has been extremely valuable, not only for my scientific growth, but also for me personally. I want to raise a toast to everyone who has been a part of this incredible journey with me. First and foremost, I am indebted to my supervisor Dr. Yuelin Zhang for his patient guidance and invaluable support during entire my graduate study. His intelligence and diligence towards research inspired me greatly. I appreciate his perceptiveness and thoughtful feedback on my research projects. I would also like to express my gratitude to my co-supervisor Dr. Xin Li, my supervisory committees Dr. George Haughn and Dr. Liang Song for their thought-provoking questions and constructive comments on my work. Their insights have greatly enriched the quality of my research projects. I appreciate the financial supports along my graduate study including China Scholarship Council, UBC TAship and Botany Department. Natural Sciences and Engineering Research Council of Canada (NSERC) provided funding to my research projects. Thanks all the current and past members of both Zhang lab and Li lab for their cooperation and warm support. I would like to thank Dr. Weijie Huang for his mentorship in genetic screening and discussion on my research projects. I also owe particular thanks to Dr. Hainan Tian and Dr. xxii Tongjun Sun who always patiently answering my endless questions and offering technical advice to me. In addition, I would like to extend my thanks to Dr. Yan Xu, Dr. Jiameng Lan, Jinyi Tan and Dr. Lei Tian. We had lots of joyful moments during the past years, like tasting cuisines from different countries, taking a short trip to nearby islands, outdoor barbecue, having parities on holidays … etc. I truly treasured the companionship with all of you and these great times will always be cherished in my life. Last but not the least, a special appreciation goes to my beloved parents. Their wholehearted love, unconditional support and constant encouragement accompanied me through the academic journey. I am also deeply grateful to my grandparents, uncles, aunts and cousins for their understanding and kindness. xxiii Dedication To my parents 1 Chapter 1: Introduction 1.1 Plant diseases All plants are subjected to diseases, as they share environments with a large repertoire of pathogenic microbes. Plant pathogens including bacteria, fungi, oomycetes, viruses and viroids. Plant disease outbreaks on major crops can lead to agricultural devastation, economic damage, and even drastic loss of human lives due to famine. The best-known example is the Irish Great Famine in the 19th century. The oomycete pathogen Phytophthora infestans caused late blight disease on potato plants, destroying three-quarters of the harvest in 1846 (Lindeboom et al., 2010). Due to the dependence on potato as a single staple food in Ireland, roughly one million people died of starvation or famine-related illness during the 1840s (Kelly, 2019). In more recent history, plant diseases in major crops (wheat, rice, maize, soybean, etc.) cause approximately 20% loss of agricultural productivity (Savary et al., 2019). As an example, coffee leaf rust, caused by fungal pathogen Hemileia vastatrix, led to a yield reduction of 16% in Central America (Boudrot et al., 2016) and tens of thousands of coffee workers losing their livelihoods (Ward et al., 2017). To better control plant diseases, it is essential to understand how the plant immune system works. 1.2 Overview of the plant immune system Plants rely on their innate immune system to respond and defend against pathogen infections. The plant immunity system is comprised of multiple complex layers. The physical and chemical barriers of plants act as the first line to impede pathogen invasion (Toruño et al., 2016). However, adapted pathogens can overcome these barriers to survive in the host apoplast. To combat these pathogens, plants deploy either cell-surface receptors to detect conserved pathogen-derived molecules, or intracellular receptors to recognize pathogen virulence factors. Activation of either 2 type of immune receptors can induce a series of effective immune responses that resist pathogen proliferation, which are referred to as Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) and Effector-Triggered Immunity (ETI) respectively (Jones & Dangl, 2006). 1.2.1 Preformed physical and chemical barriers All plants possess physical barriers to prevent pathogen invasion. The aerial surfaces of higher plants are covered by cuticle, a protective layer composed of cutin polyester and waxes (Serrano et al., 2014). The hydrophobic nature of the cuticle protects plant cells from dehydration and limits certain fungal pathogens that require standing water on the leaf surface for spore germination (Bradley et al., 2003). The plant cell wall is another physical obstacle to microbial pathogens, it has a limited pore size to restrict microbial pathogens (Underwood, 2012). Besides, upon sensing the pathogen attack, the rapid deposition of callose makes the cell wall become highly impermeable to pathogens and difficult for small insects to chew (Malinovsky et al., 2014; Sattler & Funnell-Harris, 2013). In some cases, pathogens can enter the apoplast through natural openings such as stomata. For protection against pathogen entry, plants employ sophisticated mechanisms to fine-tune stomatal closure (Melotto et al., 2017). Additionally, plants produce diverse chemical compounds to protect themselves. For example, camalexin is one of the plant secondary metabolites with antimicrobial activity. It actively accumulates in Arabidopsis thaliana upon sensing pathogen Pseudomonas syringae pv maculicola (Psm) strain ES4326 attack and it disrupts bacterial membranes to confer resistance (Rogers et al., 1996). Furthermore, plants employ antimicrobial proteins such as inducible chitinases. They accumulate in extracellular spaces where they catalyze the degradation of chitin in the cell walls 3 of pathogenic fungi (Van Den Burg et al., 2006). These preformed extracellular barriers of plants prevent the colonization of most pathogens. However, successful pathogens have strategies to bypass these physical barriers and induce innate immune responses, which are mediated by both cell surface and intracellular immune receptors. 1.2.2 PAMP-triggered immunity 1.2.2.1 PAMPs and Pattern recognition receptors Microbes share conserved protein or structural components named PAMPs, which can be detected as immunogenic signals by the plant plasma membrane localized pattern recognition receptors (PRRs). Through years of studies, a diverse set of PAMPs and their corresponding PRRs have been characterized. The Arabidopsis Flagellin-Sensitive 2 (FLS2) senses a conserved 22 amino-acid peptide flg22 of bacterial flagellin (Gómez-Gómez & Boller, 2000). EF-TU Receptor (EFR) recognizes a conserved N-terminal epitope (elf18) of the bacterial elongation factor Tu (EF-Tu) (Zipfel et al., 2006). Chitin Elicitor Receptor Kinase 1 (CERK1) detects chitin molecules from fungal cell walls (Miya et al., 2007). Plant PRRs belong to the receptor like kinase (RLK) or receptor-like protein (RLP) families (Zipfel, 2014). RLKs and RLPs are architecturally similar, both possessing an extracellular ectodomain, a transmembrane region and a cytosolic domain. Most characterized PRRs carry extracellular leucine rich repeats (LRRs), which creates an interface for protein-protein interaction or ligand-binding. For instance, flg22 binds to the concave surface spanning LRR3 to LRR16 of the FLS2 superhelical ectodomain (Schulze et al., 2010). Unlike RLKs that carry an intracellular kinase 4 domain that transduces defense signals by protein phosphorylation, RLPs only contain a short cytoplasmic tail and rely on protein partners, likely RLKs, to transmit defense signal (Altenbach & Robatzek, 2007). For example, upon perceiving chitin, RLP Chitin-elicitor binding protein (CEBiP) recruits RLK OsCERK1 to form a heterocomplex for signaling in rice (Shimizu et al., 2010). Arabidopsis Suppressor of BAK1 Interacting Receptor Kinase 1 (SOBIR1) is a common adaptor RLK for LRR-RLP type PRRs (Gust & Felix, 2014). As an example, RLP23, the receptor for nlp20 (a conserved 20-amino-acid peptide found in many Necrosis and Ethylene-Inducing Peptide 1-Like Proteins in fungi), constitutively associates with SOBIR1 (Albert et al., 2015). Upon binding of nlp20, RLP23 recruits RLK BRI1 Associated Kinase 1 (BAK1) to form a tripartite complex to initiate subsequent defense activation (Albert et al., 2015). 1.2.2.2 Activation of the PRR complexes Upon ligand perception, the PRR complex undergoes conformational changes, resulting in a series of phosphorylation events within the PRR cytoplasmic kinase domains (Liang & Zhou, 2018). In the presence of flg22, FLS2 form a heteromeric complex with BAK1, which is a common co-receptor from the Somatic-Embryogenesis Receptor-like Kinase (SERK) family. Structural analysis showed that the C-terminal segment of flg22 bridges LRRs of FLS2 and BAK1 to stabilize the heterodimerization. Meanwhile, heterodimerization of FLS2 and BAK1 leads to rapid phosphorylation of their cytoplasmic kinase domains and subsequent activation of downstream signaling events (Schulze et al., 2010). Receptor-Like Cytoplasmic Kinases (RLCKs), which lack extracellular ligand-binding domains, are essential components linking surface-localized PRRs to downstream intracellular defense 5 activities. The best characterized Arabidopsis RLCK is Botrytis-Induced Kinase 1 (BIK1). It constitutively associates with FLS2 and BAK1 at the resting state and becomes phosphorylated by BAK1 upon flg22 perception. Phosphorylated BIK1 subsequently dissociates from the PRR complex and further transphosphorylates FLS2/BAK1 to enhance signaling (Lu et al., 2010). Next, BIK1 phosphorylates downstream regulators such as Respiratory Burst Oxidase Homolog D (RbohD), Cyclic Nucleotide-Gated Channel (CNGC) proteins CNGC2 and CNGC4 to activate PTI signaling (Liang & Zhou, 2018; Zhou & Zhang, 2020). Additionally, several Arabidopsis RLCKs, including PBL1 (avrPphB Susceptible 1 (PBS1)-Like 1), BSK1 (Brassinosteroid-Signaling Kinase 1), PCRK1 (PTI Compromised Receptor like Kinase 1) and PCRK2, have been reported to play positive roles in PTI signaling by directly associating with PRR complexes (Liang & Zhou, 2018). 1.2.2.3 Defense responses downstream of PRR complexes Upon PAMP recognition, a number of cellular events are triggered to limit pathogen growth. One early PTI response is the production of reactive oxygen species (ROS). The extracellular ROS accumulates within minutes after treatment with PAMPs (Boller & Felix, 2009). In Arabidopsis, the plasma membrane localized NADPH oxidase RbohD is responsible for PAMP-triggered ROS production (Torres et al., 2005). RbohD constitutively associates with PRR complex at the plasma membrane, and it is phosphorylated by BIK1 in the presence of PAMP. ROS, such as superoxide, hydrogen peroxide and hydroxyl radical, can directly kill pathogens in the apoplast or intensify cell walls by crosslinking glycoproteins to prevent pathogen invasion (Bradley et al., 1992). On the other hand, ROS also serves as a secondary messenger to induce later responses, including activation of stomatal closure (Kwak et al., 2003). 6 Activation of PRRs also rapidly induces an influx of calcium across the plasma membrane. In Arabidopsis, heteromeric calcium-permeable channel formed by CNGC2 and CNGC4 is phosphorylated and activated by BIK1 upon pathogen attack, resulting in an increase in the concentration of cytosolic calcium (Tian et al., 2019). Calcium influx is further decoded by numerous intracellular players including calmodulins (CaMs), Calmodulin Binding Proteins (CBPs), Ca2+-dependent protein kinases (CDPKs) and other calcium binding motif-containing proteins. They interact with various components like kinases, metabolic enzymes and transcription factors to regulate immunity responses. For instance, Calmodulin-domain Protein Kinase 5 (CPK5) is activated by calcium, and it phosphorylates RbohD to regulate its activity. Additionally, RbohD-mediated ROS production also requires direct binding of Ca2+ to EF-hands in the RbohD N terminus (Kadota et al., 2015). Transcription factor CBP60g from the CBP 60 family requires CaM binding for function as a positive regulator of plant immunity (Wang et al., 2009). In plants, phosphorylation of Mitogen-Associated Protein Kinase (MAPK) cascades occurs in a couple of minutes following PRR complex activation. Three canonical MAPK cascades have been shown to play vital roles downstream of PRR complexes in regulating defense gene expression (Sun & Zhang, 2022). One consists of MAPKKK3/5-MKK4/5-MPK3/6, which can be induced by various PAMPs including flg22, nlp20 and chitin (Bi et al., 2018; Sun et al., 2018). The MEKK1-MKK1/2-MPK4 cascade is another well-studied MAPK cascade, which contributes to basal immunity against pathogens (Gao et al., 2008; Qiu et al., 2008). Recently, the MAPK cascade consisting of ANP2/3-MKK6-MPK4 was identified to be involved in plant immunity, in addition to their roles in the control of cytokinesis (Lian et al., 2018). During PTI, RLCKs function 7 downstream of PRRs, leading to activation of MAPK cascades. For instance, in the pcrk1 pcrk2 double mutant, a modest reduction of flg22-induced MAPK activation was observed (Kong et al., 2016). Upon activation, MAP kinases further induce massive transcriptional programming via phosphorylation of different transcription factors, such as WRKY33 to promote camalexin biosynthesis (Asai et al., 2002). 1.2.3 Effector-triggered susceptibility Pathogens have evolved a plethora of secreted proteins termed effectors to facilitate their colonization and undermine PTI in host plants, resulting in Effector-Triggered Susceptibility (ETS) (Jones & Dangl, 2006). Some pathogenic bacteria are able to inject effectors into plant cells through a needle-like structure such as type III secretion system (T3SS) (Grant et al., 2006), while fungal and oomycete pathogens can deliver effectors by their specialized feeding structure named haustorium or appressorium (Ribot et al., 2008). A large number of effector genes have been studied from diverse plant pathogens. Many of them contribute to virulence by targeting PRR complexes or signal transduction components of the PTI pathways to suppress the plant defense response. For example, Pseudomonas syringae (P. syringae) effector AvrPtoB functions as an E3 ligase to ubiquitinate FLS2, leading to its degradation (Göhre et al., 2008). Another effector AvrPto inhibits the kinase activities of FLS2 and EFR, thus blocking PTI responses in Arabidopsis (Xiang et al., 2008). Additionally, MAPK cascades are directly targeted by HopAI1 of P. syringae, which is a phosphothreonine lyase. HopAI1 inactivates MPK3 and MPK6 by removing the phosphate group from phosphothreonine (Zhang et al., 2007). 8 1.2.4 Effector-triggered immunity 1.2.4.1 Nucleotide-binding leucine-rich repeat receptors In addition to PRRs, higher plants have evolved a second group of immune receptors named nucleotide-binding leucine-rich repeat (NLR) proteins to specifically recognize effectors and trigger a robust and prolonged ETI response (Jones & Dangl, 2006). Typical NLRs are featured by their C terminal LRR domain that is involved in effector recognition. Their central nucleotide-binding (NB) domain serves as an oligomerization platform and ATP/ADP-binding molecular switch (Takken et al., 2006). Plant NLRs are divergent in their N-termini, which fall into two classes: TIR-NB-LRR (TNL) proteins with N-terminal Toll and Interleukin-1 (TIR)-like domains, and CC-NB-LRR (CNL) proteins with N-terminal coiled-coil (CC) domains (Meyers et al., 2003). The TIR and CC domains are known to self-associate, and they are involved in transducing immune signals after perceiving effectors (Jones et al., 2016). 1.2.4.2 Effector perception by plant NLRs Plant NLRs perceive their cognate effectors through either direct physical association or indirect recognition. In the past years, several cases of direct interaction were well characterized. In Arabidopsis, the TNL RPP1 (Recognition of Peronospora parasitica 1) directly interacts with oomycete effector ATR1 via its LRR domain, which was visualized under cryogenic electron microscopy (cryo-EM) (Krasileva et al., 2010; Ma et al., 2020). Another example of direct interaction is rice CNL Pi-ta recognizing Magnaporthe grisea effector AvrPita and their physical association was revealed by yeast two-hybrid and in vitro binding assay (Jia et al., 2000). 9 Regarding how NLRs indirectly precept effectors, three different conceptual models were proposed based on the type of components detected by NLRs. In the guard model, plant NLRs monitor the alteration of a host target (also called guardee) by pathogen effector (Van Der Hoorn & Kamoun, 2008). One of the best studied guardees is RIN4 (RPM1 Interacting Protein 4), which is localized to the plasma membrane and involved in basal resistance. P. syringae delivers effectors AvrRpt2 and AvrRpm1 to manipulate RIN4 by cleavage and phosphorylation respectively. Subsequently, the modified RIN4 is recognized by the likewise localized CNLs RPS2 (Resistance to P. syrangae 2) and RPM1 (Resistance to P. syringae pv maculicola 1), which further trigger ETI responses (Kim et al., 2005). Alternatively, in the decoy model, host targets (called decoys) serve as baits that divert effectors from the real targets and they have lost their original functionality in the absence of its cognate NLRs (Van Der Hoorn & Kamoun, 2008). As an example, the effector AvrPphB from P. syringae cleaves host cytoplasmic kinase PBS1 and PBLs which induces CNL RPS5-mediated immune responses (Ade et al., 2007). Unlike PBLs which are positive regulators of PTI, PBS1 has no critical role in PTI and loss of PBS1 does not benefit bacterial colonization in plants. Thus, PBS1 likely has evolved as a decoy that mimics effector targets to trap the pathogen into a recognition event (Van Der Hoorn & Kamoun, 2008). As an extension of the decoy model, integrated decoy model implies the recognition of effectors by paired co-acting NLRs. Some NLR proteins had evolved with integrated decoy (ID) domains at their C-termini, which resemble effector targets and can trigger immune responses by the second NLR upon effector binding (Cesari et al., 2014). Examples include Arabidopsis TNL pair RRS1 10 (Resistance to Ralstonia solanacearum 1) and RPS4 (Recognition of Peronospora parasitica 4). RRS1 carries a C-terminal ID WRKY DNA-binding domain which can be targeted by bacterial effector AvrRPS4 or PopP2. Interaction of effector AvrRps4 or PopP2 with the WRKY domain of RRS1 triggers immune responses by preventing the activation of defense related WRKY transcription factors and releasing RPS4 from RPS4/RRS1 heterodimer to form a RPS4 homodimer, further transducing defense signals (Narusaka et al., 2009). 1.2.4.3 NLRs-mediated immune responses Perception between effectors and their cognate NLRs leads to NLR activation, which involves NLR protein conformational change and oligomerization. The biochemical and structural studies on Arabidopsis CNL HopZ-Activated Resistance 1 (ZAR1) revealed that ZAR1 forms a wheel-like pentamer (called resistosome) and potentially inserts itself into the plasma membrane upon bacterial effector AvrAC perception (Wang et al., 2019). Recently, activated ZAR1 resistosomes was shown to possess calcium-permeable cation channel activity to initiate immunity and cell death (Bi et al., 2021). By contrast, tetramerization of TIR domains of TNLs forming the final active resistosome with NADase holoenzyme activity is a common activation mechanism for TNLs, such as Arabidopsis RPP1 and Nicotiana benthamiana (N. benthamiana) ROQ1 (Recognition Of XopQ 1) (Ma et al., 2020; Martin et al., 2020). Many CNLs, such as RPS2 and RPM1, require an integrin-like protein Non Race-Specific Disease Resistance 1 (NDR1) anchored in the plasma membrane for immune activation (Aarts et al., 1998). In parallel, signaling activated by TNLs converges on the lipase-like proteins Enhanced Disease 11 Susceptibility 1 (EDS1) and its analogue Phytoalexin Deficient 4 (PAD4) or Senescence Associated Gene 101 (SAG101) (Dongus & Parker, 2021; Wiermer et al., 2005). Upon recognition of pathogen effectors, the NADase holoenzyme activity of TIRs catalyze the production of small molecules, pRib-AMP/ADP and ADPr-ATP/di-ADPR, which activate EDS1-PAD4 and EDS1-SAG101 heterodimers. Subsequently, activated heterodimers induce assembly of CNL-type helper NLRs, Activated Disease Resistance 1 (ADR1) and N Requirement Gene 1 (NRG1) resistosomes as Ca2+ channels to mediate disease resistance and cell death (Song et al., 2024). Similar to PTI, the output of ETI also including ROS burst, MAPK cascade activation, and transcriptional reprogramming. However, ETI is more amplified and faster than PTI, and ETI is often associated with the hypersensitive response (HR), a type of localized cell death that serves to restrict growth of biotrophic pathogens and their spread (Jones & Dangl, 2006). 1.3 PTI and ETI crosstalk PTI and ETI used to be considered as two largely independent branches of the plant immune system. Recently, emerging experimental evidence supports the intricate crosstalk between PTI and ETI, which synergistically enhance each other to prime a robust defense response (Ngou et al., 2021; Pruitt et al., 2021; Tian et al., 2021; Yuan et al., 2021). As mentioned previously, common signaling components including RbohD, MAP Kinase, CNGC calcium channels are shared by both PTI and ETI. The maximal phosphorylation of RbohD relies on PTI signaling, while ETI signaling rapidly augments the expression level of RbohD (Ngou et al., 2021; Yuan et al., 2021). HR cell 12 death is compromised in the fls2 efr1 cerk1 mutant, implying that PTI potentiates ETI outputs such as HR to halt pathogen proliferation. Furthermore, hNLRs contribute to PTI potentiation (Tian et al., 2021; Pruitt et al., 2021). The upregulation of PTI responses, including early defense gene expression as well as SA accumulation, was impaired in the eds1/pad4/sag101 and hNLR adr1s/nrg1s mutants in Arabidopsis (Pruitt et al., 2021; Tian et al., 2021). Protein-protein interactions were even detected between EDS1, PAD4, ADR1s and the common adaptor RLK SOBIR1 for PTI (Pruitt et al., 2021). Many TIR genes are also induced by PAMP treatment to boost PTI responses. Interestingly, such induction requires RLCKs such as PCRK1/2 and PBL19/20 but is independent of EDS1 (Tian et al., 2021). 1.4 Systemic acquired resistance Locally induced PTI and ETI activation can further induce a secondary immune response termed systemic acquired resistance (SAR) in the distal parts of the host plant. SAR can confer long-lasting and broad-spectrum resistance against subsequent pathogen attack (Durrant & Dong, 2004). Description of the SAR phenomenon can be traced back to 1960s, where tobacco plants challenged with tobacco mosaic virus (TMV) developed increased resistance against tobacco necrosis virus (TNV) and other bacterial pathogen infections in distal tissues (Ross, 1961). Later, in the 1970s, establishment of SAR was shown to be associated with the induction of Pathogenesis Related (PR) genes. PR proteins accumulate locally and systemically in tobacco in response to TMV (Gianinazzi & Kassanis, 1974; Van & Van Kammen, 1970). Nowadays, PR genes are widely used as molecular markers for both local defense and SAR. 13 1.4.1 Signaling molecules in SAR Establishment of SAR involves the generation of defense signals at inoculated tissue as well as their long-distance transportation via phloem to the uninfected distal tissues, which was supported by grafting experiment (Dean & Kuć, 1986; White, 1979). Which mobile signal accounts for long-distance communication in SAR has been a subject of controversy for many years. Several metabolites such as salicylic acid (SA), methyl salicylate, azelaic acid, glycerol-3-phosphate, the amino acid-derivative pipecolic acid (Pip) and its derivative N-hydroxypipecolic acid (NHP) have been suggested as candidate mobile signals (Shah et al., 1997). Amongst them, the role of SA and NHP in SAR responses have been studied most extensively as their effects are the most pronounced. SA emerged as a signaling molecule in SAR during the early 1990s. Pathogen infection leads to drastic increase of SA levels in both local and systemic plant tissues (Malamy et al., 1990). SA accumulation was also shown to be essential for SAR in studies using transgenic plants expressing the SA-degrading enzyme salicylate hydroxylase from bacteria (Gaffney et al., 1993). Exogenous application of SA or its analogue 2,6-dichloroisonicotinic acid (INA) is sufficient to induce SAR (Cao et al., 1994). Later studies on Arabidopsis mutants defective in SA biosynthesis or perception further confirmed the importance of SA in SAR (Delaney et al., 1995; Nawrath & Métraux, 1999). Despite its vital role in SAR, SA is unlikely a long-distance signal based on early data from grafting experiments that the chimeric tobacco containing a wild type scion and SA-deficient rootstock was still able to develop SAR (Vernooij et al., 1994). NHP and its biosynthetic precursor Pip were lately identified as two critical metabolites involved in SAR establishment (Hartmann & Zeier, 2019; Tian & Zhang, 2019). Blocking Pip or NHP 14 biosynthesis results in SAR impairment. In turn, exogenously supplied NHP was sufficient to induce SAR in wild-type plants (Chen et al., 2018; Hartmann et al., 2018). Moreover, tracking translocation of the exogenously applied NHP together with several other observations strongly suggest that NHP travels, after accumulating in the locally inoculated leaves, to distant leaves in the course of SAR induction (Hartmann et al., 2018; Holmes et al., 2019; Schnake et al., 2020). 1.5 Biosynthesis and regulation of SA and NHP 1.5.1 SA biosynthesis in plants Plants use two independent routes to produce SA, the isochorismate synthase (ICS) and phenylalanine (Phe) ammonia-lyase (PAL) pathways (Dempsey et al., 2011; Hartmann & Zeier, 2019; Huang et al., 2020), both initiating in chloroplast from the primary metabolite chorismate, a product of the shikimate pathway. The ICS pathway characterization started with the study of Arabidopsis sid2 (salicylic acid induction deficient 2) mutant, which carries a mutation in ICS1 (Wildermuth et al., 2001). Arabidopsis genome carries two ICS genes, ICS1 and ICS2. ICS1 contributes to about 90% of total SA accumulation induced by pathogens while ICS2 only plays a minor role (Garcion et al., 2008). Both ICS1 and ICS2 are localized to the chloroplast, suggesting that the conversion of chorismate to isochorismate occurs in the chloroplast (Garcion et al., 2008; Wildermuth et al., 2001). Studies of another SA induction deficient mutant sid1 revealed that mutations in the MATE transporter Enhanced Disease Susceptibility 5 (EDS5) lead to greatly decreased SA levels and enhanced disease susceptibility to bacterial pathogens (Nawrath et al., 2002). EDS5 localizes to the chloroplast envelop. It facilitates the transport of isochorismate from plastids to the cytosol 15 (Rekhter et al., 2019). Lastly, avrPphB Susceptible 3 (PBS3) serves as an amidotransferase that catalyzes the conjugation of glutamate to isochorismate in the cytosol (Torrens-Spence et al., 2019). The resulted product isochorismate-9-glutamate (IC-9-Glu) can spontaneously decay to yield SA. Enhanced Pseudomonas Susceptibility 1 (EPS1), an enzyme processing pyruvoyl-glutamate lyase activity, can further boost the production of SA from IC-9-Glu in addition to the spontaneous reaction (Torrens-Spence et al., 2019). The PAL pathway was shown to contribute to SA biosynthesis in a number of plant species, such as Arabidopsis, rice, soybean, and tobacco (Huang et al., 2010; Shine et al., 2016; Xu et al., 2017; Yalpani et al., 1993). Isotope labeling experiments revealed that SA can be synthesized from Phe via transcinnamic acid (CA) and benzoic acid (BA) in tobacco (Ribnicky et al., 1998; Yalpani et al., 1993). The conversion of Phe to CA by PAL is one of the rate-determining steps in SA biosynthesis (Vogt, 2010). In Arabidopsis, loss of all four PAL genes resulted in a 75% reduction of basal SA production and 50% decrease in pathogen-induced SA accumulation, which genetically confirmed that PAL are not only involved in basal SA production, but also contribute to pathogen-induced SA biosynthesis (Huang et al., 2010). Abnormal Inflorescence Meristem 1 (AIM1) mediates the conversion of CA to BA. AIM1 encodes a hydroxyacyl-CoA hydrolyase involved in beta-oxidation (Richmond & Bleecker, 1999), which is required for the production of benzoylated metabolites in Arabidopsis seeds (Bussell et al., 2014). A putative BA 2-hydroxylase (BA2H) was proposed to hydroxylate BA to SA (Leon et al., 1995), but the encoding gene of BA2H has not been identified yet. 16 1.5.2 Transcriptional regulation of SA biosynthetic genes As SA over accumulation has a detrimental effect to plant fitness, plants maintain relatively low SA levels during normal growth and development. Pathogen infections usually lead to a rapid increase in SA levels. Such dynamic changes in SA levels are a result of tight regulation of SA production. In Arabidopsis, many transcription factors are involved in regulating genes involved in SA biosynthesis. Pathogen-induced ICS1 expression and SA biosynthesis are mainly controlled by two plant-specific transcription factors, SAR-Deficient 1 (SARD1) and CaM-Binding Protein 60 g (CBP60g) (Wang et al., 2009, 2011; Zhang et al., 2010). Loss of both SARD1 and CBP60g leads to almost complete block of the induction of ICS1 and SA accumulation by bacterial pathogens (Wang et al., 2011; Zhang et al., 2010). Moreover, the induction of EDS5 and PBS3 by the bacterial pathogen Psm ES4326 is greatly reduced in the sard1 cbp60g double mutant, suggesting that genes involved in the pathogen-induced SA biosynthesis pathway are coordinately regulated by SARD1 and CBP60g. In the promoter of ICS1, there is a typical Teosinte branched 1/Cycloidea/PCF (TCP)-binding site (Wang et al., 2015). Yeast one-hybrid analysis showed that transcription factors TCP8 and TCP9 bind to this site, at which they function redundantly in promoting ICS1 expression and SA biosynthesis (Wang et al., 2015). Transcription factors NTM1-Like 9 (NTL9) and CCA1 Hiking Expedition (CHE) were also shown to directly regulate ICS1 expression through physical interaction (Zheng et al., 2015). NTL9 promotes ICS1 expression and SA biosynthesis in stomatal immunity (Zheng et al., 2015). CHE is involved in the regulation of circadian oscillations of ICS1 and SA levels as well as induction of ICS1 expression and SA accumulation in distal tissue during SAR (Zheng et al., 2015). 17 Several WRKY transcription factors have been implicated in positive regulation of SA biosynthesis. WRKY28 and WRKY75 directly bind to the promoter of ICS1 to promote its expression and SA production (Guo et al., 2017; van Verk et al., 2011). In addition, overexpression of WRKY46 in Arabidopsis protoplasts resulted in increased ICS1 expression (van Verk et al., 2011). The induction of ICS1 expression by Pseudomonas syringae pv. tomato (Pst) DC3000 avrRpm1, avrRpt2 and avrB, but not Pst DC3000 is greatly reduced in wrky8 and wrky48 mutant plants, suggesting that the effector-induced ICS1 expression is largely dependent on WRKY8 and WRKY48 (Gao et al., 2013). However, whether WRKY8 and WRKY48 directly regulate ICS1 is still unclear. WRKY transcription factors are also involved in the negative regulation of SA biosynthetic genes. WRKY18 and WRKY40 were shown to directly target ICS1, EDS5, and PBS3, and negatively regulate their expression (Birkenbihl et al., 2017). Besides, in the wrky54 wrky70 double mutant, the basal level as well as the Psm ES4326 avrRpt2 induced expression level of ICS1 are much higher than in wild type plants, demonstrating their negative role in regulating SA production (Wang et al., 2006). Two ethylene signaling components Ethylene Insensitive 3 (EIN3) and EIN3-Like 1 (EIL1) exert a negative role in SA biosynthesis by repressing ICS1. EIN3 binds to the promoter of ICS1 and the ein3-1 eil1-1 double mutant displayed elevated ICS1 and SA levels in the absence of pathogen attack (Chen et al., 2009). CBP60a is another transcription factor implicated in negative regulation of SA biosynthesis (Truman et al., 2013). In cbp60a mutant plants, there is an increase in basal 18 ICS1 and SA levels, suggesting that CBP60a either directly or indirectly affects the expression of ICS1 and SA biosynthesis. 1.5.3 SA perception and downstream signaling SA is perceived by two types of receptors, Non-Expressor of Pathogenesis-Related Proteins (NPR1) and its paralogs NPR3/NPR4, which regulate SA-induced defense gene expression and pathogen resistance (Cao et al., 1994; Zhang et al., 2006). The npr1 loss-of-function mutant is compromised in both SAR and basal resistance (Cao et al., 1997), whereas the npr3 npr4 double mutant shows enhanced resistance against virulent bacterial and oomycete pathogens (Zhang et al., 2006). A gain-of-function mutation in NPR4 (named npr4-4D) leads to constitutive repression of SA-induced immune responses and compromised basal defense when challenged with the virulent bacteria Psm ES4326 (Ding et al., 2018). NPR1, NPR3 and NPR4 all interact with a subgroup of redundant basic leucine zipper transcription factors TGA2, TGA5 and TGA6 (TGACG-Binding Factors) (Zhang et al., 1999, 2006), but play different roles in transcriptional regulation of SA-induced downstream gene expression. NPR1 functions as a transcriptional co-activator, whereas NPR3/NPR4 act as transcriptional co-repressors. As the SA level elevates during pathogen infection, the transcriptional repression activities of NPR3/NPR4 are inhibited by SA while the transcriptional activation activity of NPR1 is promoted by SA. Upon SA-binding, both derepression of NPR3/4 and activation of NPR1 lead to strong immune activation through downstream transcription factors including SARD1 and WRKY70 (Ding et al., 2018). 19 1.5.4 NHP biosynthesis Studies on Arabidopsis SAR-deficient (sard) mutants elucidated that NHP is synthesized from a three-step metabolic pathway in plants. The first committed step of NHP biosynthesis is catalyzed by the aminotransferase AGD2-Like Defense response protein 1 (ALD1). It uses lysine as a substrate and catalyzes its conversion to 𝜖 -amino- 𝛼 -keto caproic acid, which cyclizes spontaneously to form 1- piperideine-2-carboxylic acid (P2C) (Ding et al., 2016; Hartmann et al., 2017). Then SARD4 catalyzes the reduction of P2C to Pip, an immediate precursor of NHP (Ding et al., 2016; Hartmann et al., 2017). The full Pip pathway can be reconstituted by introducing just ALD1 and SARD4 into Escherichia coli (E.coli) (Ding et al., 2016). The formation of NHP from Pip requires Flavin-dependent Monooxyenase 1 (FMO1). Biochemical analysis revealed that FMO1 catalyzes the N-hydroxylation of Pip, yielding NHP (Chen et al., 2018; Hartmann et al., 2018). NHP exists as both free and glycosylated forms. Glycosyltransferase UDP-dependent glycosyltransferase 76b1 (UGT76B1) is responsible for the conversion of NHP to NHP-O-glycoside (Bauer et al., 2021; Cai et al., 2021; Holmes et al., 2021). 1.5.5 Transcriptional regulation of NHP biosynthetic genes Similar to SA, NHP biosynthetic genes are coordinately regulated by SARD1 and CBP60g in Arabidopsis. Chromatin immunoprecipitation (ChIP) analysis revealed that SARD1 and CBP60g also target NHP synthetic genes ALD1, SARD4, and FMO1 (Sun et al., 2015; Sun et al., 2018). In the sard1 cbp60g double mutant, the induction of ALD1, SARD4, and FMO1 by Psm ES4326 is greatly reduced. On the other hand, overexpression of SARD1 leads to increased ALD1 and SARD4 expression and elevated Pip levels (Sun et al., 2018). In addition, Pip and NHP levels following infection by Psm ES4326 are much lower in the sard1 cbp60g double mutant than in wild-type 20 plants (Sun et al., 2018; Sun et al., 2020), suggesting that SARD1 and CBP60g activate Pip and NHP biosynthesis through upregulation of their biosynthetic genes. WRKY33 is another transcription factor that positively regulates Pip and NHP biosynthesis (Wang et al., 2018). In wrky33 mutant plants, Pst DC3000 avrRpt2-induced ALD1 and FMO1 expression, as well as Pip accumulation, is dramatically reduced. ChIP-PCR analysis revealed that WRKY33 binds to the promoter of ALD1, suggesting that it directly regulates the expression of ALD1 to promote Pip and NHP biosynthesis. However, loss of WRKY33 results in elevated SA levels during Botrytis cinerea infection (Birkenbihl et al., 2012). It is therefore possible that WRKY33 may indirectly promote NHP accumulation by affecting SA level. 1.6 SARD1 and CBP60g SARD1 and CBP60g are two closely related transcription factors, they coordinately modulate pathogen-induced expression of SA and NHP biosynthetic genes through direct binding to their promoters (Sun et al., 2015; Wang et al., 2009, 2011; Zhang et al., 2010). Following pathogen infection, the expression of SARD1 and CBP60g is rapidly induced (Wang et al., 2009) and these two transcription factors are recruited to ICS1 promoter to activate its expression and SA biosynthesis (Zhang et al., 2010). Both SAR and local resistance are hardly attenuated in sard1 and cbp60g single mutants, while sard1 cbp60g double mutants exhibit severely compromised local and systemic resistance, suggesting SARD1 and CBP60g play additive roles in basal resistance and SAR (Zhang et al., 2010). Notably, ChIP analysis revealed that SARD1 and CBP60g also target the promoters of many defense-related genes in addition to SA and NHP biosynthetic genes. Other candidate targets include genes encoding components of PRR complex such as BAK1 21 and BIK1, genes in MAPK cascades such as MEKK1 and MPK3, and genes required for ETI such as EDS1 and PAD4 (Sun et al., 2015). 1.6.1 Regulation of SARD1 and CBP60g SARD1 and CBP60g belong to the CBP60 transcription factor family. Both carry a DNA binding domain in their conserved central regions (Zhang et al., 2010). However, SARD1 and CBP60g have diverse N- and C- terminal regions and only share 39% identity at the amino acid sequence level, implying that their activities may be differentially regulated. Indeed, overexpression of SARD1 leads to constitutive activation of defense responses, which were not observed in transgenic plants overexpressing CBP60g (Zhang et al., 2010). CBP60g carries a 45 amino-acid CaM binding domain (CBD) at its N terminus. It was shown that CBP60g requires CaM binding for its function in disease resistance and PAMP-induced SA accumulation (Wang et al., 2009). However, SARD1 does not have a similar N-terminal motif as in CBP60g, and it cannot bind CaM (Wang et al., 2009). Transcription factors TGA1 and TGA4 are involved in the induction of SARD1 and CBP60g (Sun et al., 2018). In the tga1 tga4 double mutant, the induction of SARD1 and CBP60g, as well as SA and Pip accumulation, are greatly reduced at the early stage of plant defense. ChIP-PCR analysis revealed that TGA1 binds to the promoter of SARD1 but not CBP60g, suggesting that the TGAs directly regulate SARD1 and indirectly regulate CBP60g expression. Another transcription factor GTL1 was shown to directly regulate pathogen-induced CBP60g expression (Völz et al., 2018). GTL1 binds to the promoter region of CBP60g, and the expression level of CBP60g is significantly reduced in the gtl1 mutant after flg22 treatment. Recent studies showed that CBP60b, another 22 member of the CBP60 family, also directly regulates SARD1 expression. Over-expression of CBP60b leads to elevated SARD1 expression and constitutive defense responses. In addition, transient express CBP60b in N. benthamiana can activate the expression of the pSARD1::luciferase reporter gene (Huang et al., 2021). In addition, several transcription factors, including WRKY70 and calmodulin binding transcription activators CAMTA1/CAMTA2/CAMTA3, are involved in the negative regulation of SARD1 and CBP60g expression. WRKY70 binds to a GACTTTT motif in the SARD1 promoter, and basal SARD1 expression level is elevated in wrky70 mutant plants, suggesting that it may inhibit SARD1 expression in the absence of pathogen infection (Zhou et al., 2018). In the camta3-1 knockout mutant, SA level is elevated and the expression levels of SARD1 and CBP60g are dramatically increased at low temperature (Du et al., 2009; Kim et al., 2013). On the other hand, the induction of SARD1 and CBP60g by flg22 and Psm ES4326 is significantly reduced in the gain-of-function camta3-3D mutant (Jacob et al., 2018; Sun et al., 2020). CAMTA3 can bind to the promoter of CBP60g, but not SARD1, in planta, suggesting that it directly regulates CBP60g expression, and its effect on SARD1 is most likely indirect (Sun et al., 2020). The close homologs of CAMTA3, CATMA1 and CAMTA2, have partially redundant functions with CAMTA3 (Kim et al., 2013). The elevated SARD1 and CBP60g expression, and autoimmune phenotypes in camta3-1, are dramatically enhanced in the camta1 camta2 camta3 triple mutant. 1.7 SNC2-mediated immune pathway Arabidopsis Suppressor of npr1-1, constitutive 2 (SNC2) encodes an RLP that is required for basal resistance against pathogens (Zhang, et al., 2010). Loss-of-function mutations in SNC2 result in 23 enhanced susceptibility to the pathogenic bacteria Pst DC3000. Conversely, a gain-of-function mutation in the conserved GXXXG motif of the SNC2 transmembrane domain leads to constitutively activated defense responses. The autoimmune mutant snc2-1D exhibits a dwarf morphology, increased levels of PR gene expression and SA accumulation. snc2-1D also confers enhanced resistance against the virulent oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) Noco2 and the bacterial pathogen Pst DC3000 (Zhang, et al., 2010). From a suppressor screen of snc2-1D npr1-1 designed to search for immune regulators downstream of SNC2, several bda (BDA: Bian Da; becoming big in Chinese) mutants were identified. BDA1 encodes an ankyrin-repeat containing transmembrane protein (Yang et al., 2012), while WRKY70 is a transcription factor (Zhang, et al., 2010). As mutations in WRKY70 block the constitutive defense responses in snc2-1D npr1-1, WRKY70 likely functions in an NPR1-independent defense pathway downstream of SNC2 (Zhang, et al., 2010). BDA1 functions upstream of NPR1 and WRKY70, as the gain-of-function bda1-17D mutant with a single amino acid change in the second transmembrane domain activates both NPR1-dependent and WRKY70-dependent defense responses (Yang et al., 2012). In addition, bda4-1D, also known as npr4-4D, suppresses the constitutive defense responses in snc2-1D npr1-1 and the mutation in NPR4 is responsible for the suppression of the snc2-1D npr1-1 mutant phenotypes. npr4-4D suppresses the expression of SARD1, CBP60g and WRKY70, which are required for the autoimmunity of snc2-1D npr1-1 (Ding et al., 2018). Recent studies showed that SARD1 and CBP60g are two regulators in the SNC2-mediated defense pathway and are required for the autoimmune responses in the snc2-1D mutant (Sun et al., 2015). 24 The cbp60g-1 snc2-1D and sard1-1 snc2-1D double mutants display slight suppression of the dwarfism of snc2-1D, while the cbp60g-1 sard1-1 snc2-1D triple mutant exhibits wild type like morphology. Quantitative analysis indicated that the elevated levels of defense marker gene expression and SA accumulation in snc2-1D are partially suppressed in the double mutants and completely suppressed in the triple mutant. Additionally, the enhanced resistance against Hpa. Noco2 in snc2-1D is slightly reduced in the double mutants and almost lost in the triple mutant. As mutations in SARD1 and CBP60g result in additive suppression of the autoimmunity of snc2-1D, SARD1 and CBP60g function in two parallel pathways downstream of SNC2. Figure 1.1 SNC2-mediated defense signaling. Transcription factor SARD1 and CBP60g define two parallel pathways downstream of SNC2. 25 1.8 Thesis objectives As introduced above, SARD1 and CBP60g are two master transcription factors in immune regulation that directly control a large number of defense gene expression. However, how SARD1 and CBP60g are activated by the immune signals from plasma membrane receptors remain unclear. The identification of SARD1 and CBP60g as parallel critical regulators of SA biosynthesis and snc2-1D-mediated autoimmunity allows the SARD1-dependent signaling pathway and the CBP60g-dependent signaling pathway to be studied separately. To identify the components that are involved in the CBP60g-dependent signaling pathway downstream of SNC2, a suppressor screen was conducted on sard1-1 snc2-1D double mutant background and suppressor mutants have been isolated. Mutation in components functioning downstream of SNC2 in the CBP60g dependent signaling pathway would result in the suppression of sard1-1 snc2-1D’s autoimmunity. My thesis goal is to clone the causal suppressor genes and elucidate how they contribute to plant immunity regulation. Studies of the mutants from the sard1-1 snc2-1D suppressor screen will facilitate the identification of upstream regulators of CBP60g and help us better understand how RLP-mediated immunity and SA signaling pathway are regulated. 26 Chapter 2: The clathrin adaptor ECA4 regulates plant immunity through modulating BDA1 levels 2.1 Summary Plants deploy plasma membrane localized pattern recognition receptors (PRRs) to sense pathogens and activate downstream defense responses. One class of plant PRRs are receptor-like proteins. In Arabidopsis, activation of RLP SUPPRESSOR OF NPR1-1, CONSTITUTIVE 2 (SNC2) signals through parallel transcription factors SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60g (CBP60g). The constitutive defense responses of snc2-1D were almost fully suppressed in cbp60g-1 sard1-1 snc2-1D, while mutation in CBP60g or SARD1 has limited effects on the autoimmunity of snc2-1D. Here, we report the identification of a clathrin adaptor ECA4 as an inhibiting factor in SNC2-mediated defense signaling. A gain-of-function mutation eca4-4D suppresses the autoimmunity of sard1-1 snc2-1D. In addition, similar to eca4-4D single mutant, overexpression of ECA4 exhibit compromised resistance against pathogens, indicating its negative role in plant immunity. Interestingly, transient expression of ECA4 or ECA4-4D in Nicotiana benthamiana reduces the protein levels of BDA1, a transmembrane protein required for SNC2-mediated immunity. Consistently, eca4-4D partially suppresses the autoimmunity of bda1-17D. Taken together, opposite to the roles of clathrin and its homologous clathrin adaptor EPSIN1 that are required for PRR endocytosis, ECA4 appears to suppress immunity through modulating the protein levels of BDA1. 27 2.2 Introduction In nature, plants encounter pathogenic microbes comprising viruses, bacteria, fungi and oomycetes. Successful sensing and defending against microbial infections are essential to their survival. Through plant–pathogen co-evolution, plants have evolved sophisticated surveillance mechanisms to effectively ward off pathogenic intruders. In plants, pattern recognition receptors (PRRs) localized on the plasma membrane act as initial sensors to recognize conserved pathogen associated molecular patterns (PAMPs), resulting in pattern-triggered immunity (PTI) (Monaghan & Zipfel, 2012). PTI is featured by the rapid deployment of profound physiological changes in plant cells, including but not limited to the activation of mitogen-activated protein kinase (MAPK) cascades, reactive oxygen species (ROS) burst, calcium influx, callose deposition, defense hormone salicylic acid (SA) production and transcriptional reprogramming (Zhou & Zhang, 2020). All known plant PRRs are receptor like kinases (RLKs) or receptor like proteins (RLPs) (Zipfel, 2014). Both RLKs and RLPs possess an extracellular ligand-binding region and a transmembrane domain, while RLPs are distinguished from RLKs by their lack of a cytoplasmic kinase domain. The Arabidopsis genome encodes 57 putative RLPs (Wang et al., 2008). SUPPRESSOR OF NPR1, CONSTITUTIVE 2 (SNC2) is one of the identified RLPs found to be required for basal resistance against bacterial pathogens (Zhang, et al., 2010). Loss of SNC2 in Arabidopsis leads to enhanced susceptibility against virulent bacteria strain Pseudomonas syringae pv tomato (Pst) DC3000. The gain-of-function snc2-1D mutant, which bears a substitution of the second Gly for Arg in the conserved GXXXG motif of the transmembrane helix, exhibits a dwarfed morphology and constitutively activated immunity (Zhang, et al., 2010). From a suppressor screen of snc2-1D npr1-1, BDA1 (for Bian Da; “becoming big” in Chinese) was identified as an essential regulator of 28 SNC2-mediated immunity (Yang et al., 2012). BDA1 carries five ankyrin-repeats at its N-terminus and four transmembrane domains at its C-terminus. Loss of BDA1 can completely suppress the small stature and constitutive immunity in snc2-1D npr1-1 mutant, leading to increased susceptibility to pathogens. SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN (CaM)-BINDING PROTEIN 60g (CBP60g) are two plant specific transcription factors that exert broad roles in the regulation of plant immunity (Huang et al., 2020). Upon pathogen infection, the expression levels of SARD1 and CBP60g are rapidly induced (Wang et al., 2009). They can directly bind to the promoters of genes that control SA biosynthesis, such as ISOCHORISMATE SYNTHASE 1 (ICS1), ENHANCED DISEASE SUSCEPTIBILITY 5 (EDS5), and AVRPPHB SUSCEPTIBLE 3 (PBS3) (Sun et al., 2015; Wang et al., 2011). The sard1 cbp60g double mutant exhibit almost complete loss of pathogen induced SA accumulation and severe enhanced susceptibility (Wang et al., 2011; Zhang et al., 2010), revealing their essential roles in immune regulation. Both SARD1 and CBP60g are members of the CBP60 transcription factor family, they share similarities in the central DNA binding domain but vary in N- and C- termini. CBP60g can bind CaM in vitro (Wang et al., 2009), but SARD1 lacks the N terminal CaM binding motif. Moreover, overexpression of SARD1 can induce plant defense responses, while transgenic lines over-expressing CBP60g exhibit a wild type-like phenotype (Zhang et al., 2010). Thus, SARD1 seems to be regulated mainly through its transcriptional control, while CBP60g is more likely modulated by Ca2+ and CaM. 29 SARD1 and CBP60g are required for SNC2-mediated immunity. The autoimmune phenotypes of snc2-1D are partially suppressed by the single mutant of sard1 or cbp60g, but completely suppressed by sard1 cbp60g double mutant (Sun et al., 2015), suggesting SARD1 and CBP60g define two parallel pathways that regulate defense responses downstream of SNC2. Chromatin immunoprecipitation (ChIP)-seq analyses revealed that CBP60g and SARD1 directly bind to the promoters of a large number of defense regulatory components to orchestrate plant immunity (Sun et al., 2015). However, the components that function downstream of SNC2 leading to activation of SARD1 and CBP60g are largely unknown. Upon pathogen challenge, proper subcellular distribution and adequate amounts of the PRRs are critical for defense signaling activation and proper immune output. After ligand binding, PRRs undergo endocytosis and endosomal trafficking for signaling attenuation (Claus et al., 2018). Clathrin-mediated endocytosis (CME) is a major endocytic mechanism for the internalization of plasma membrane proteins (Narasimhan et al., 2020). In recent years, CME has been reported to contribute to the turnover and activity of plant pattern recognition receptors including EF-TU RECEPTOR (EFR), the danger peptide PEP RECEPTOR 1 (PEPR1), and LYSM-CONTAINING RECEPTOR-LIKE KINASE 5 (LYK5) (Erwig et al., 2017; Mbengue et al., 2016; Ortiz-Morea et al., 2016). CME happens in the order of nucleation, cargo selection, clathrin coat assembly, membrane scission, and vesicles uncoating (McMahon & Boucrot, 2011). The endocytic adaptor-related TPLATE complex (TPC) has been reported to play a pivotal role in the initial nucleation step. The eight core subunits of TPC (TPLATE, TASH3, LOLITA, TWD40-1, TWD40-2, TML, AtEH1, and AtEH2) assemble at sites of CME, preceding the recruitment of adaptor protein 30 complex 2 (AP2), clathrin, and dynamin-related proteins. Loss of TPC function is detrimental to CME and plant survival (Gadeyne et al., 2014). To identify the components involved in the CBP60g-dependent signaling pathway downstream of SNC2, a suppressor screen was conducted on sard1-1 snc2-1D double mutant background. Here we report the identification and characterization of a suppressor of sard1-1 snc2-1D, which carries a gain-of-function mutation, eca4-4D, in the clathrin adaptor protein ECA4. ECA4 appears to be involved in regulating SNC2-mediated immune responses by modulating the protein level of BDA1. 2.3 Material and methods 2.3.1 Plant materials and growth conditions All Arabidopsis mutants used are in the Columbia (Col-0) ecotype. The sard1-1 snc2-1D, bda1-17D mutants were reported previously (Sun et al., 2015; Yang et al., 2012). eca4-4D was isolated from a cross between the original suppressor mutant 125-1 and Col-0. eca4-1 (salk_114631c) and cap1-3 (SAIL_418_A09) are exonic T-DNA insertion alleles. eca4-5 and cap1-4 are deletion mutants generated by CRISPR/Cas9 genome editing. The two independent eca4 cap1 double mutants were obtained by crossing eca4-1 with cap1-3 and crossing eca4-5 with cap1-4 respectively. Unless specified otherwise, plants were grown on soil under a long-day condition of 16/8-hour light/dark regime at 22 ºC. Arabidopsis seedlings for RNA extraction and qRT-PCR were grown on half-strength Murashige and Skoog (½ MS) medium under long-day condition for 12 days. 31 Plants for SA measurement and pathogen infection assays were grown on soil under a short-day condition of 8/16-hour light/dark regime at 22 ºC for 4 weeks. 2.3.2 Generation of deletion mutants The CRISPR/Cas9 system was used for generating eca4-5 and cap1-4 deletion mutants. Genomic sequences of ECA4 and CAP1 were subjected to CRISPR-PLANT (http://www.genome.arizona. edu/crispr/CRISPRsearch.html) to select the guide RNAs. The selected guide RNAs were evaluated with Cas-OFFinder (http://www.rgenome.net/cas-offinder/). Golden Gate cloning was used to introduce guide RNAs into the CRISPR binary vector pHEE401 according to previously published protocols (Xing et al., 2014; Wang et al., 2015). Arabidopsis plants were transformed with the Agrobacterium carrying the derived plasmid by floral dip method (Clough & Bent, 1998). T1 plants were analyzed for deletion by PCR. Later, homozygous deletion mutants were obtained in the T2 generation. Primers used for generating the CRISPR constructs and genotyping the deletion mutants are listed in Table 2.1. 2.3.3 Mutagenesis and suppressor screen Ethyl methane sulfonate (EMS) mutagenesis was carried out with a previously described protocol (Li & Zhang, 2016). About 5,000 sard1-1 snc2-1D mutant seeds were soaked with 20 mM EMS solution for 16h with gentle shaking to induce point mutations. After washing with 100 mM Na2S2O3 three times, the mutagenized seeds were sterilized with 15 % bleach containing 0.1 % Tween 20 and plated on ½ MS plates. M1 seedlings were transplanted to soil after 10 days- and grown to maturity. In M2 generation, plants were directly grown on soil and screened for plants with larger size than the parental sard1-1 snc2-1D mutant. 32 2.3.4 Mapping-by-sequencing Mapping-by-sequencing was performed as previously described (Sun et al., 2020). The suppressor mutants were backcrossed with sard1-1 snc2-1D plants and plants segregating for the dwarf and suppressor like phenotypes in the F2 progeny were identified. The homozygosity of suppressor-like F2 lines was further confirmed in the F3 generation. Leaf tissue from about 50 homozygous F3 progeny were pooled together and subjected to DNA extraction. The bulk genomic DNA was sent for whole genome sequencing (WGS). By comparing with the TAIR10 reference genome, the ratios of single nucleotide polymorphisms (SNPs) were calculated and used to identify the linkage region. Genes containing nonsynonymous mutations in the linkage region were selected as candidate genes for knockout analysis. 2.3.5 Gene expression analysis For each sample, approximately 50 mg plant tissue from 4 individual 12-day-old seedlings of the indicated genotypes were collected. Three biological replicates were analyzed for each genotype. RNA was isolated using Plant RNA Mini-Preps Kit (Bio Basic, Canada) and reverse transcribed to cDNA with OneScript Reverse Transcriptase (Abcam, Canada). Quantitative reverse transcript-PCR (qRT-PCR) analysis of the cDNA samples was carried out with the SYBR Premix Ex Taq II (Takara, Japan). The primers used for qPCR were reported previously (Sun et al., 2015). Transcripts were normalized to ACTIN1 and then compared with that of wild type plants. 2.3.6 Pathogen infection assays For Hyaloperonospora arabidopsidis (Hpa) Noco2 infection, two-week-old seedlings were sprayed with conidiospores suspension at a concentration of 5x104 spores per mL water. Plants 33 were subsequently covered with a transparent lid and transferred to a growth chamber at 18°C under a 12-h-day/12-h-night cycle with 80% humidity. Sporulation was quantified at 7 days post inoculation by counting conidia spores with a hemocytometer. 4-6 individual plants were pooled as a single sample. 2.3.7 SA extraction and quantification The procedure of SA extraction and measurement was modified from a published protocol (Li et al., 1999). About 100 mg of plant tissue from 3-4 individual plants was collected as one sample and ground into fine powder with liquid nitrogen. Samples were extracted first with 0.6 mL 90% methanol and were subsequently vortexed and sonicated for 20 min. After spinning at 13,000 rpm for 10 min, the supernatant was collected, and the pellet was re-extracted with 0.4 mL of 100% methanol as above. The supernatants from the two extractions were combined and dried by vacuum. Afterwards, 0.5 mL of 5% (w/v) trichloroacetic acid was added to the dry samples and the samples were vortexed and sonicated for 5 min. Following centrifugation at 13,000 rpm for 10 min, the supernatant was collected and extracted three times with 0.5 mL of extraction medium (ethylacetate/cyclopentane/isopropanol:100/99/1 v/v/v). Each time, after spinning at 13,000 rpm for 1 min, the top organic phases were collected. The combined organic phases were then dried by vacuum. The samples were next resuspended in 0.5 mL of mobile phase (0.2M KAc, 0.5mM EDTA, pH=5.0 with acetic acid) by vortexing and sonicating for 5 min. After the final spinning at 13,000 rpm for 5 min, the supernatants were collected and subject to high-performance liquid chromatography (HPLC) to determine the amount of SA. 34 2.3.8 Transient expression in Nicotiana benthamiana For transient expression assays in Nicotiana (N.) benthamiana, leaves of about 4-week-old plants were infiltrated with Agrobacterium carrying the indicated construct combinations. The final OD600 for ECA4-FLAG, ECA4-4D-FLAG and empty vector was 0.8. The OD600 value for BDA1-HATurboID was 0.2. Two days after infiltration, about 80 mg tissue from the infiltrated area was collected and grinded into fine powder with liquid nitrogen. Then samples were homogenized in 2 × SDS loading buffer and subjected to immunoblotting with either 𝛼-HA or 𝛼-FLAG antibodies. Protein abundance was quantified using ImageJ (https://imagej.nih.gov/ij/). Table 2.1 Primers used in Chapter 2 Primer name DNA sequence (5’ to 3’) At2g25430-DT1-BsFF0 ATATATGGTCTCGATTGGCGAGATCGTCTGAGTATCGTTTTAGAGCTAGAAATAGC At2g25430-DT2-BsRR0 ATTATTGGTCTCGAAACGGTAAGGGTAAAGCTACGCCAATCTCTTAGTCGACTCTAC At2g25430-dele-F GAAAAGTGGTGTGTCGGTGA At2g25430-dele-R CGAATGGGTCTTGGTTCACT At2g25430-homo-F GAAGCTCCTCCTCCGGTAGT At2g25430-tDNA-F TGATTTTCGCGATGAAGCGC At2g25430-tDNA-R CCAAACTGGCTTGACCTCTC AT4G32285-DT1-BsFF0 ATATATGGTCTCGATTGGTAGTAGTCACCAAAGTAAGTTTTAGAGCTAGAAATAGC AT4G32285-DT2-BsRR0 ATTATTGGTCTCGAAACTCCCAAGCTCTGCTGCTGGCAATCTCTTAGTCGACTCTAC AT4G32285-dele-F AAGAAGACGCGTGACTGGAT AT4G32285-dele-R CTGAGGTGCTCACGTGTTGT AT4G32285-homo-F ATTTGCAGAGGCTGCTTGAT AT4G32285-tDNA-F CGCTAATAACGGAAAATGGG 35 AT4G32285-tDNA-R GATGTTCCAAATTTCAATTGG At2g25430-ATG-KpnI-F CCGGGGTACCATGGCTCCGAGTATTCGAAAAG At2g25430-CDS-BamHI-R CGCCGCGGATCCGTAAGGATTGTTGTAGTAATAC At2g25430-pro-KpnI-F CCGGGGTACCGTCTATCAATTTGCAGGAACG 2.4 Results 2.4.1 Identification and characterization of an EMS suppressor of sard1-1 snc2-1D To identify regulators of CBP60g-dependent defense signaling in SNC2-mediate immunity, an EMS-mutagenesis-based forward genetic screen was performed on the sard1-1 snc2-1D mutant. Any plants with larger size than sard1-1 snc2-1D were selected for further analysis. 125-1 was one of the mutants identified from the screen. In this suppressor mutant, the autoimmunity-related small size of sard1-1 snc2-1D was almost completely suppressed (Figure 2.1A). qRT-PCR analysis showed that the elevated expression of PATHOGENESIS-RELATED (PR) genes PR1 (Figure 2.1B) and PR2 (Figure 2.1C) in sard1-1 snc2-1D is fully suppressed in the 125-1 mutant. In addition, 125-1 displayed significantly reduced level of defense hormone SA compared with sard1-1 snc2-1D (Figure 2.1D). The enhanced resistance against oomycete pathogen Hpa Noco2 observed in sard1-1 snc2-1D is also blocked in 125-1 (Figure 2.1E). Taken together, the 125-1 mutant suppresses the dwarf morphology as well as constitutive defense responses of sard1-1 snc2-1D. 36 Figure 2.1 Identification and characterization of the sard1 snc2-1D suppressor mutant 125-1. (A) Morphology of three-week-old soil-grown plants of the indicated genotypes under long-day condition. Scale bar is 1 cm. (B and C) Relative expression levels of PR1 (B) and PR2 (C) in the indicated genotypes. Transcript levels were normalized with ACTIN1. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (D) Free SA levels in the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (E) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). 2.4.2 Mapping‑by‑sequencing of 125-1 To identify the causal mutation leading to the suppression of sard1-1 snc2-1D phenotypes, mapping-by-sequencing was performed. The 125-1 mutant was first backcrossed with sard1-1 snc2-1D. The F1 progeny exhibits a similar morphology as 125-1 (Figure 2.2A), indicating that 125-1 carries a dominant mutation. Further, a 163:56 segregation of wild type-like and sard1-1 37 snc2-1D -like plants was observed in the F2 generation, consistent with the expected 3:1 ratio for a single dominant nuclear mutation (χ2 = 0.0381; P value = 0.8453). F3 seeds from the wild type-like F2 plants were harvested and planted to identify the homozygous suppressors. Leaf tissue from 46 homozygous F3 progeny exhibiting wild type-like phenotype were pooled together and subjected to DNA extraction and WGS. Comparison with the reference genome sequence revealed a linkage region on chromosome 2 (Figure 2.2B). A nonsynonymous G-to-A mutation in the second exon of At2g25430 with SNP frequency of 1 attracted our attention. The same mutation had previously been identified as being responsible for the suppression of autoimmunity of snc2-1D npr1-1 eds5-3 (unpublished data from Dr. Yuli Ding). Therefore, At2g25430 was selected as a prime candidate gene for knockout analysis. Figure 2.2 Mapping-by-sequencing of mutant 125-1. (A) Morphologies of 24-day-old soil-grown plants of the indicated genotypes under long-day condition. Scale bar is 1 cm. 38 (B) Linkage map showing the linkage of 125-1 mutation to the middle of chromosome 2. The G to A substitution in At2g25430 is highlighted with a red data point. 2.4.3 Deletion mutants of At2g25430 in 125-1 restore the constitutively activated immunity To examine whether the mutation in At2g25430 is responsible for the suppression of sard1-1 snc2-1D autoimmunity phenotypes, CRISPR/Cas9 was used to generate At2g25430 knock out mutants in the original 125-1 mutant background. Two independent CRISPR lines were recovered with 586-bp deletions inside At2g25430 (Figure 2.3A), and both displayed even smaller size than that of sard1-1 snc2-1D (Figure 2.3B). In addition, the CRISPR lines restored the constitutive expression of defense marker genes as well as the elevated SA accumulation (Figure 2.3C-E). Consistently, these deletion lines showed enhanced disease resistance against Hpa Noco2 similar to the sard1-1 snc2-1D mutant line (Figure 2.3F). Therefore, loss of this dominant mutation in the original suppressor mutant can relieve the suppression of sard1-1 snc2-1D autoimmunity phenotypes, suggesting that the mutation in At2g25430 is responsible for the 125-1 defects. Furthermore, transforming the mutant gene At2g25430G1069A under its native promoter (np::At2g25430G1069A) into the sard1-1 snc2-1D background suppresses the dwarfism of sard1-1 snc2-1D (Figure 2.4A), confirming the causal relationship between the mutation in At2g25430 and the suppressor phenotypes of 125-1. 39 Figure 2.3 CRISPR deletion alleles of At2g25430 in the 125-1 background restore the autoimmunity of sard1 snc2-1D. (A) Mutations in At2g25430. 125-1 carries a G to A substitution in At2g25430 (top). #3 and #11 are two independent deletion lines of At2g25430 in the 125-1 mutant, deleting the same genomic region (bottom). (B) Morphologies of 24-day-old soil-grown plants of the indicated genotypes under long-day condition. Scale bar is 1 cm. (C and D) Expression levels of PR1 (B) and PR2 (C) in the indicated genotypes as normalized by ACTIN1. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (E) Free SA levels in the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (F) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). 40 2.4.4 eca4-4D carries a gain-of-function mutation in ECA4. To determine whether At2g25430G1069A is a gain-of-function or dominant-negative mutation, At2g25430 was overexpressed under 35S promoter in sard1-1 snc2-1D background. The resulting transgenic lines show a wild-type size (Figure 2.4A). Consistently, these overexpression transgenic lines exhibit a similar susceptibility against Hpa Noco2 as the original suppressor mutant 125-1(Figure 2.4B), suggesting that At2g25430G1069A is a gain-of-function mutation. At2g25430 was previously named as ECA4 (EPSIN-LIKE CLATHRIN ADAPTOR), eca4-4D will hence be referred to ECA4 carrying a gain-of-function mutation. By crossing 125-1 with Col-0 plants, an eca4-4D single mutant was isolated. When ECA4 was overexpressed under the 35S promoter in Col-0 background, the transgenic lines are more susceptible than wild type plants (Figure 2.4C), suggestive a negative role of ECA4 in immunity. ECA4 shares high sequence similarity (89.3%) with its homolog CLATHRIN ASSOCIATED PROTEIN 1 (CAP1). Both CAP1 and ECA4 are clathrin adaptor proteins that may be involved in CME (Gadeyne et al., 2014). Upon Hpa Noco2 infection, in contrast to the result from the eca4-4D mutant, two independent lines of eca4 cap1 double mutants are more resistant than wild type plants (Figure 2.4C). This further confirmed that eca4-4D carries a gain of function mutation. Furthermore, as shown in Figure 2.5D, while eca4 cap1 double mutants supported less Hpa Noco2 sporulation, the Hpa Noco2 growth in eca4 and cap1 single mutants was similar to that in wild type plants, indicating that ECA4 and CAP1 function redundantly in terms of plant immunity regulation. 41 Figure 2.4 eca4-4D carries a gain-of-function mutation in ECA4. (A) Morphologies of 24-day-old soil-grown plants of the indicated genotypes under long-day condition. Scale bar is 1 cm. (B) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Transgenic lines of 35S::At2g25430 and np::At2g25430G1069A are in the sard1-1 snc2-1D background. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (C) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (D) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 4). 2.4.5 Overexpression of ECA4 or eca4-4D reduces BDA1 protein levels As a related component of the TPLATE complex that function in CME, ECA4 likely contributes to the internalization and activity of plasma membrane proteins. Thus, we tested whether eca4-4D mutation affects the ankyrin-repeat transmembrane protein BDA1 protein levels. A binary vector 42 carrying 35S::ECA4-3FLAG, 35S::ECA4-4D-3FLAG or empty vector (EV-3FLAG) was transiently transformed, together with BDA1-HATurboID, into N. benthamiana. As shown in Figure 2.5A and B, co-expression of ECA4-3FLAG with BDA1-HATurboID resulted in decreased BDA1 levels in N. benthamiana. Besides, co-infiltration of ECA4-4D-3FLAG with BDA1-HATurboID also causes less BDA1 protein level (Figure 2.5C and D). Therefore, overexpression of ECA4 or eca4-4D negatively affects BDA1 protein stability and ECA4 may participate in the turnover of protein BDA1. Figure 2.5 Overexpression of ECA4 or eca4-4D reduces BDA1 protein levels in N. benthamiana. (A) Immunoblot analysis of protein levels of BDA1-HATurboID in the absence or presence of ECA4-3FLAG in N. benthamiana. Agrobacterium carrying plasmids expressing BDA1-HATurboID together with EV-3FLAG or ECA4-3FLAG were co-infiltrated into one half of each N. benthamiana leaf. Samples were collected at 48 h post-inoculation (hpi). Equal loading is shown by Ponceau S staining of a non-specific band. 43 (B) Quantification of relative band intensity of (A). The intensity of BDA1-HATurboID bands upon co-infiltration with EV-FLAG was set as 1. Error bars represent standard deviations. *** indicates statistical differences (P < 0.05, unpaired t test; n = 6). (C) Immunoblot analysis of protein levels of BDA1-HATurboID in the absence or presence of ECA4-4D-3FLAG in N. benthamiana. Agrobacterium carrying plasmids expressing BDA1-HATurboID together with EV-3FLAG or ECA4-4D-3FLAG were co-infiltrated into one half of each N. benthamiana leaf. Samples were collected at 48 hpi. Equal loading is shown by Ponceau S staining of a non-specific band. (D) Quantification of relative band intensity of (C). The intensity of BDA1-HATurboID bands upon co-infiltration with EV-FLAG was set as 1. Error bars represent standard deviations. **** indicates statistical differences (P < 0.05, unpaired t test; n = 6). 2.4.6 eca4-4D partially suppress the autoimmunity of bda1-17D Previously we identified bda1-17D, a gain-of-function allele of BDA1 with a single amino acid change in the second transmembrane domain resulting in the constitutive activation of cell death and defense responses (Yang et al., 2012). bda1-17D displayed elevated levels of PR gene expression as well as SA accumulation. Consistently, bda1-17D showed enhanced resistance against Hpa Noco2 challenge. As shown in Figure 2.6A, eca4-4D bda1-17D double mutant is wild-type like, and the up-regulations of PR1 and PR2 expression in bda1-17D were largely blocked (Figure 2.6B and C). In addition, the elevated accumulation of SA and enhanced resistance against Hpa Noco2 in bda1-17D was also partially affected by eca4-4D (Figure 2.6D and E). Therefore, eca4-4D partially suppress the autoimmunity of bda1-17D. 44 Figure 2.6 eca4-4D partially suppress the autoimmunity of bda1-17D. (A) Morphologies of four-week-old soil-grown plants of the indicated genotypes under short-day condition. Scale bar is 1 cm. (B and C) Expression levels of PR1 (B) and PR2 (C) in the indicated genotypes as normalized by ACTIN1. RNA was extracted from 21-day-old soil-grown plants under short-day conditions. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (D) Free SA levels in the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). 45 (E) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 4). 2.5 Discussion From a suppressor screen of sard1-1 snc2-1D, we identified a gain-of-function allele of ECA4. Overexpression of ECA4 in Col-0 background results in enhanced susceptibility against Hpa Noco2. In addition, overexpression of ECA4 and eca4-4D both negatively affect the protein levels of BDA1, a transmembrane protein required for SNC2-mediated immunity. The constitutively activated defense responses of a gain-of-function allele bda1-17D were partially suppressed by eca4-4D. These observations suggest that ECA4 is a negative regulator of immunity, and it is likely to function through modulating the protein level of BDA1. ECA4 is a member of the ANTH/ENTH/VHS superfamily, which serves as mediators of vesicle membranes, cargoes and clathrin during the endomembrane vesicles’ formation process. It carries a single folded N-terminus AP180 N-terminal homology (ANTH) domain for binding with lipids of the membrane and a C-terminus unstructured region that interacts with clathrin and with the folded domains of other coat components (Owen et al., 2004). Although the gain-of-function E357K mutation that we identified here does not harbor the ANTH domain or any known short peptide motif, E357 is conserved in CAP1 and another two members of the ANTH subfamily, PICALM5a and PICALM5b (PHOSPHATIDYLINOSITOL BINDING CLATHRIN ASSEMBLY PROTEIN). The homologous PICALM5 pair has been reported to control the pollen tube tip localization of ANXUR receptor kinases (Muro et al., 2018). Whether E357 is part of an uncharacterized short motif that mediates interactions with clathrin and the ear domain of the large subunits of heterotetrameric adaptor protein complexes (APs) remains to be studied. 46 ECA4 contributes to the cargo recycling from the trans-Golgi Network/Early Endosome (TGN/EE) to the plasma membrane. The role of ECA4 in recycling brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) as well as RESPIRATORY BURST OXIDASE HOMOLOG D (RbohD) has been validated in eca4 mutants (Lee et al., 2022; Nguyen et al., 2018). However, the role of ECA4 in endomembrane trafficking, and in particular in CME, is not well understood. ECA4 and CAP1 share a remarkably high sequence homology and they were speculated to have redundant functions. Here we provide evidence of their function redundancy in regulating plant immunity, as the growth of Hpa Noco2 in eca4, cap1, and wild type plant is comparable, but the eca4 cap1 double mutants support less growth of the Hpa Noco2. ECA4 and CAP1 localized to the TGN/EE and plasma membrane (Nguyen et al., 2018), and both interact with TML, one of the core components of TPLATE complex, which might function in the CME of plasma membrane-based proteome (Gadeyne et al., 2014). As overexpression of ECA4 and ECA4-4D gain-of-function mutation leads to decreased of BDA1 protein level, it is possible that BDA1 was internalized to the TGN/EE and are then transported to the vacuole for degradation, resulting in reduced protein levels at the plasma membrane. It remains to be determined whether ECA4 and CAP1 are involved in endocytosis of BDA1 to endosomes. Clathrin protein, composed of light and heavy chains, mediates the endocytosis of PRRs into a common endosomal pathway, therefore executing an important role in the regulation of plant immunity. In Arabidopsis, the clathrin heavy chain knockout mutant chc2-1 was defective in flg22-induced stomata closure, callose deposition and resistance against bacterial pathogen (Mbengue et al., 2016). Similarly, the knock down of six clathrin heavy-chain genes in a N. benthamiana 47 transient expression system resulted in a significant decrease in FLAGELLIN SENSING2 (FLS2) and PEPR1-containing endosomes (Mbengue et al., 2016). There are two clathrin-binding motifs in the C-terminal, unstructured region of ECA4 and CAP1, FS motif (472FS and 452FS in ECA4 and CAP1, respectively) and DPF motif (574DPF and 555DPF in ECA4 and CAP1, respectively) (Barth & Holstein, 2004). The close homologs of ECA4 and CAP1, including ECA1, EpsinR1 and EpsinR2 bind to clathrin (Song et al., 2006; Song et al., 2012). Recently, ECA4 and CAP1 were shown to associate with clathrin (Wang et al., 2023), but how ECA4 and CAP1 serve as clathrin adaptor proteins to contribute to clathrin-related process requires further investigation. 48 Chapter 3: The pre-mRNA splicing factor ZOP1 contributes to plant immunity regulation 3.1 Summary The CALMODULIN-BINDING PROTEIN 60g (CBP60g) and its homolog SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) are two master transcription factors of plant immunity. They define two parallel pathways downstream of the receptor like protein SUPPRESSOR OF NPR1-1, CONSTITUTIVE 2 (SNC2). Autoimmune responses in the gain-of-function snc2-1D mutant are partially blocked by either sard1 or cbp60g single mutation, but completely suppressed by the sard1 cbp60g double mutant. Here, we report the identification and characterization of ZOP1, a pre-mRNA splicing factor that functions as a positive regulator of SNC2-mediated immunity. Loss-of-function mutations in ZOP1 suppress the autoimmunity of sard1-1 snc2-1D. zop1 single mutants exhibit compromised resistance against pathogens. RNA-seq analysis on zop1 mutant revealed that a large number of defense response genes are downregulated, including BDA1, encoding an essential regulator of SNC2-mediated defense pathway. Overall, our study suggests that the pre-mRNA splicing factor ZOP1 contributes to plant immunity regulation through modulating the transcript levels of defense response genes. 3.2 Introduction Plants rely on their innate immune system to perceive and word off pathogen threats. Plants utilize cell surface localized pattern-recognition receptors (PRRs) to sense the conserved pathogen-associated molecular patterns (PAMPs), leading to the activation of pattern triggered immunity (PTI) (Boller & Felix, 2009). Adapted pathogens can deliver effectors to interfere with PTI, 49 leading to effector triggered susceptibility (ETS) (Jones & Dangl, 2006). In turn, plants have evolved intracellular resistance (R) proteins to recognize specific pathogen effectors and activate robust effector triggered immunity (ETI), which usually leads to localized hypersensitive cell death response (HR) (Belkhadir et al., 2004). Some PRRs belong to the receptor like protein (RLP) family. A typical RLP has an extracellular leucine-rich repeat domain, a transmembrane motif, and a tiny cytoplasmatic tail (Zipfel, 2014). Arabidopsis RLP SNC2 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE 2) plays an essential role in basal resistance and PTI (Yang et al., 2012; Zhang, et al., 2010). Loss of SNC2 results in compromised resistance against pathogenic bacteria Pseudomonas syringae pv tomato (Pst) DC3000 as well as non-pathogenic bacteria Pst DC3000 hrcC-. On the other hand, a gain-of function mutation in the conserved GXXXG motif of the SNC2 transmembrane domain leads to constitutively activated defense responses. This snc2-1D mutant has smaller size, accumulates high levels of defense hormone salicylic acid (SA), constitutively expresses PATHOGENESIS-RELATED (PR) genes, and shows enhanced pathogen resistance to oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) Noco2 (Zhang, et al., 2010). The plant-specific transcription factor CALMODULIN BINDING PROTEIN 60 g (CBP60g) and its close homolog SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) are two master transcription factors in plant immunity (Zhang et al., 2010). They coordinately regulate the expression of SA biosynthetic genes including ISOCHORISMATE SYNTHASE 1 (ICS1), ENHANCED DISEASE SUSCEPTIBILITY 5 (EDS5) and AVRPPHB SUSCEPTIBLE 3 (PBS3) during pathogen infection (Sun et al., 2015; Wang et al., 2011; Zhang et al., 2010). In the sard1 50 cbp60g double mutant, there is a dramatic decrease of SA accumulation following bacterial infection (Wang et al., 2011; Zhang et al., 2010). Chromatin-immunoprecipitation-sequencing (ChIP-seq) analysis further revealed that SARD1 and CBP60g directly modulate the expression of many key regulators in PTI and ETI responses, indicating that they play broad roles in the regulation of plant immunity (Sun et al., 2015). SARD1 and CBP60g function downstream of SNC2 and contribute to the autoimmunity of snc2-1D (Sun et al., 2015). The autoimmune responses of snc2-1D were partially blocked in sard1-1 snc2-1D and cbp60g-1 snc2-1D double mutants and almost completely abolished in the sard1-1 cbp60g-1 snc2-1D triple mutant. Consistently, the dwarfism of snc2-1D was slightly attenuated in sard1-1 snc2-1D and cbp60g-1 snc2-1D, whereas cbp60g-1 sard1-1 snc2-1D was wild type-like. Therefore, SARD1 and CBP60g function in parallel and define two independent pathways that orchestrate SNC2-mediated immunity. However, how the plasma membrane localized SNC2 transduces signals to activate SARD1 or CBP60g remains obscure. Alternative splicing of mRNA precursors expands the transcript diversity in eukaryotes. Pre-mRNA splicing is performed by the spliceosome, a dynamic protein complex containing more than 100 proteins and 5 small nuclear ribonucleoprotein particles (snRNP) (Shang et al., 2017). In Arabidopsis, many splicing factors have been identified, such as ZOP1 (ZINC-FINGER AND OCRE DOMAIN-CONTAINING PROTEIN 1), STA1(STABILIZED1, PRP6-LIKE SPLICING FACTOR) and RDM16 (RNA-DIRECTED DNA METHYLATION 16) (Du et al., 2015; Huang et al., 2013; Zhang et al., 2013) Multiple studies have shown that many splicing factors are not only components of the spliceosome that participate in pre-mRNA splicing, they also regulate 51 different biological processes. Among them, ZOP1 was shown to function in pre-mRNA splicing, transcriptional gene silencing, and cold stress response (Du et al., 2015; Zhang et al., 2013). However, its role in plant defense responses is unclear. To identify regulators required for CBP60g-dependent defense responses downstream of SNC2, a suppressor screen was performed in the sard1-1 snc2-1D background. Here, we report the identification and characterization of suppressor 116-1. The 116-1 mutant contains a mutation in ZOP1. Further analysis showed that ZOP1 is involved in regulating SNC2-mediated immune responses by affecting the transcript level of BDA1 (for Bian Da; “becoming big” in Chinese), a key component downstream of SNC2. 3.3 Materials and Methods 3.3.1 Plant materials and growth conditions All Arabidopsis plants used in this study are in the Columbia (Col-0) ecotype. The sard1-1 snc2-1D mutants was reported previously (Sun et al., 2015). A CRISPR construct targeting ZOP1 was generated as previously described in chapter 2. For transgene complementation, the genomic DNA fragment of ZOP1 driven by its native promoter was PCR-amplified from wild-type genomic DNA and cloned into pBasta-3HA vector. Primers used for generating the constructs and genotyping the deletion mutants are listed in Table 3.1. All plants used in this study were grown under a long-day condition of 22°C and 16 h light/8 h dark cycles unless specifically mentioned. 52 3.3.2 Mutant Screening Ethyl methane sulfonate (EMS) mutagenesis was performed following a previously described protocol (Li & Zhang, 2016). The sard1-1 snc2-1D mutant seeds were treated with EMS. About 40,000 M2 progeny from 2000 M1 plants were grown on soil and screened for plants with increased plant size compared with the sard1-1 snc2-1D mutant. 3.3.3 Mapping-by-sequencing Mapping-by-sequencing was performed as previously described (Sun et al., 2020). The 116-1 mutant was backcrossed with sard1-1 snc2-1D plant. 39 of total 169 F2 segregants that showed larger size were kept to F3 generation. Leaf tissue from 36 confirmed larger homozygous plants were pooled together and used for DNA extraction. The resulting genomic DNA was sequenced by Illumina whole genome sequencing (WGS). The frequency of SNPs was calculated using Excel and used to identify the linkage region and candidate mutations. 3.3.4 Characterization of mutants For SA quantification, plants were grown on soil under a short-day condition of 8/16-hour light/dark regime at 22 ºC for 4 weeks. SA was extracted from 100 mg of leaf tissue and measured by high-performance liquid chromatography (HPLC) using a previously described protocol (Li et al., 1999). To analyze the resistance to Hpa Noco2, 2-week-old seedlings were sprayed with Hpa Noco2 spores at a concentration of 50,000 spores per mL of water. Plants were grown for another 7 days 53 in a chamber at 18°C under a 12-h-day/12-h-night cycle with 80% humidity before counting the number of spores with a hemocytometer. 3.3.5 Gene Expression and Splicing Pattern Analysis Arabidopsis seedlings for RNA extraction were grown on half-strength Murashige and Skoog (½ MS) medium under long day condition for 12 days. RNA was extracted using the Plant RNA Mini-Preps Kit (Bio Basic, Canada) and treated with RQ1 RNAase-free DNAase (Promega, Canada) to eliminate genomic DNA contamination. Reverse transcription reactions were carried out with OneScript Reverse Transcriptase (Abcam, Canada). Quantitative real-time PCR was performed using the SYBR Premix Ex Taq II (Takara, Japan). Primers used for amplification of PR1, PR2, and ACTIN1 were described previously (Zhang et al., 2003). The primers for analyzing the transcription level and the intron retention events of BDA1 are included in Table 3.1. 3.3.6 RNA-seq analysis For RNA-sequencing (RNA-seq), 12-day-old seedlings of wild type and zop1-2 grown on ½ MS media were collected. RNA extraction and RNA-seq were carried out using services provided by Smartgenomics Technology Institute (Tianjin, China). 39~62 million raw reads were checked for quality, and low-quality reads, adapter sequences, and contaminations were filtered out. The clean reads of each sample were aligned to the publicly available reference genome of Arabidopsis (TAIR10) using Hierarchical Indexing for Spliced Alignment of Transcripts 2 (HISAT2) (Kim et al., 2019). Transcript assembly and quantification were performed using StringTie (Pertea et al., 2015). GO analysis was completed using the statistical enrichment test from the Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/). For 54 intron-retention analysis, replicate Multivariate Analysis of Transcript Splicing (rMATS) tool was used to identify reliable expressed introns and adjust P-value < 0.01 were regarded as intron-retention events (Shen et al., 2014). Table 3.1 Primers used in Chapter 3. Primer name Primer sequence (5’ to 3’) At1g49590-DT1-F ATATATGGTCTCGATTGGTATTAGAAACCATGACCTGTTTTAGAGCTAGAAATAGC At1g49590-DT2-R ATTATTGGTCTCGAAACCTCCTTTCACAGCTCTTTTCAATCTCTTAGTCGACTCTAC At1g49590-dele-F TTCGCATCTTTTCTCTCCAAA At1g49590-dele-R AGCAGCTTCTCTTGCCTTCA At1g49590-homo-F GGTGCACCAGAGGATGGTA At1g49590-pro-KpnI-F CCGGGGTACCCAGAGAATGACGCAAAACCA At1g49590-SpeI-R CGGCTAGACTAGTAAAGGGTCTATTGTAAAGAC BDA1-RT-F CGTGGTAGAGGAGGTATGACAC BDA1-RT-R GCATCCACCCTGTGAGAACT BDA1-splicing-F1 CAGAAAATGCGCGATAGTGA BDA1-splicing-R1 AGAGAGCCTCCGGTCTTACC BDA1-splicing-F2 TTTGGCAATGGAACTGATGA BDA1-splicing-R2 TCACTATCGCGCATTTTCTG 3.4 Results 3.4.1 Identification and characterization of suppressor mutant 116-1 To better understand CBP60g-dependent signaling pathway downstream of SNC2, a forward genetic screen was performed to identify mutants with suppressed autoimmunity of sard1-1 snc2-1D. Considering the small size of sard1-1 snc2-1D plants, the mutagenized population was screened for mutants with larger stature than sard1-1 snc2-1D. 116-1 was one of the suppressor 55 mutants recovered. As shown in Figure 3.1A, 116-1 has an intermediate size between the wild type and sard1-1 snc2-1D. It displayed reduced expression of the defense marker gene PR1 and PR2 compared to the sard1-1 snc2-1D mutant (Figure 3.1B and C). sard1-1 snc2-1D was previously reported to accumulate higher SA (Sun et al., 2015), and the SA level in 116-1 was considerably lower than in sard1-1 snc2-1D (Figure 3.1D). Consistently, 116-1 also supported more growth of the virulent oomycete pathogen Hpa Noco2 compared to sard1-1 snc2-1D (Figure 3.1E). Thus 116-1 shows suppression of all aspects of the autoimmune phenotypes of sard1-1 snc2-1D. Figure 3.1 Identification and characterization of the sard1 snc2-1D suppressor mutant 116-1. (A) Morphology of three-week-old soil-grown plants of the indicated genotypes under long-day condition. Scale bar is 1 cm. (B and C) Relative expression levels of PR1 (B) and PR2 (C) in the indicated genotypes. Transcript levels were normalized with ACTIN1. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (D) Free SA levels in the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). 56 (E) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). 3.4.2 Mapping‑by‑sequencing of 116-1 When 116-1 was backcrossed to sard1-1 snc2-1D, the resulting F1 plants appeared sard1-1 snc2-1D like, indicating that 116-1 carries a recessive mutation (Figure 3.2A). In the F2 progeny, a 130:39 segregation of suppressor-like and sard1-1 snc2-1D -like plants was observed, agreeing with the expected 3:1 ratio for a single recessive nuclear mutation (χ2 = 0.3521; P value = 0.5637). Leaf tissue from the 36 confirmed homozygous F3 populations displayed no segregation of plants with sard1-1 snc2-1D-like phenotype was collected for DNA extraction and WGS. Upon data analysis, a linkage region was identified on Chromosome 1 (Figure 3.2B). In this region, one of the candidate genes At1g49590 (encoding ZOP1) carries a C109T mutation located in the second exon, leading to a premature stop codon (Figure 3.3A). Figure 3.2 Mapping-by-sequencing of 116-1. 57 (A) Morphologies of 24-day-old soil-grown plants of the indicated genotypes under long-day condition. Scale bar is 1 cm. (B) Linkage map showing the linkage region of the 116-1 mutation on chromosome 1. The mutation in ZOP1 is highlighted using a red data point. 3.4.3 Loss of ZOP1 suppresses the autoimmunity of sard1-1 snc2-1D To determine whether the mutation identified in ZOP1 is responsible for suppressing sard1-1 snc2-1D, a CRISPR/Cas9 construct targeting the candidate gene ZOP1 was generated and transformed into the sard1-1 snc2-1D background. Two independent CRISPR lines with large deletions in ZOP1 displayed a larger plant size (Figure 3.3B). The constitutive expression of PR1 and PR2 of sard1-1 snc2-1D was largely suppressed in CRISPR lines (Figure 3.3C and D). Consistently, the elevated SA accumulation as well as enhanced disease resistance against Hpa Noco2 in sard1-1 snc2-1D was abolished in the CRISPR deletion lines (Figure 3.3E and F). Thus, we conclude that loss-of-function mutations in ZOP1 suppresses sard1-1 snc2-1D. 58 Figure 3.3 Deletion mutants of ZOP1 in sard1-1 snc2-1D suppress its autoimmunity. (A) Mutations in ZOP1 (At1g49590). 116-1 carries a C to T substitution in At1g49590, leading to a premature stop codon (top). #3 and #48 are two independent CRISPR deletion lines of ZOP1 in the sard1-1 snc2-1D background, deleting the same genomic region (bottom). (B) Morphology of 24-day-old soil-grown plants of the indicated genotypes under long-day condition. Scale bar is 1 cm. (C and D) Expression levels of PR1 (C) and PR2 (D) in the indicated genotypes as normalized by ACTIN1. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (E) Free SA levels in the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (F) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). Further, when a construct expressing ZOP1 driven by its native promoter, np::ZOP1-3HA, was transformed into 116-1, the plant size of transgenic lines was reverted back to that as sard1-1 snc2- 59 1D (Figure 3.4A). Also, the transgenic lines restore the enhanced disease resistance against Hpa Noco2 (Figure 3.4B). These data further support the hypothesis that the mutation in ZOP1 in 116-1 causes the autoimmunity of sard1-1 snc2-1D. Figure 3.4 np::ZOP1 complements the defects of the suppressor 116-1. (A) Morphology of 21-day-old soil-grown plants of the indicated genotypes under long-day condition. #1 and #2 are two independent complementation lines of ZOP1 in the 116-1 background. Scale bar is 1 cm. (B) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). 3.4.4 zop1 single mutants exhibit compromised resistance against pathogen zop1 single mutants were generated using CRISPR/Cas9 to study the role of ZOP1 in plant immunity. Three independent zop1 deletion alleles, 13#, 15# and 24# (hereafter zop1-1, zop1-2 and zop1-3), show similar stature as wild type plants (Figure 3.5A). To determine whether disease resistance is affected in the zop1 mutants, Hpa Noco2 was inoculated on wild type and the zop1 60 mutants. As shown in Figure 3.5B, zop1 mutants supported significantly more Hpa Noco2 growth than wild type plants, indicative of a positive role of ZOP1 in plant immunity. Figure 3.5 Compromised immunity in the zop1 mutants. (A) Morphology of 21-day-old soil-grown plants of the indicated genotypes under long-day condition. Scale bar is 1 cm. (B) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Scale Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). 3.4.5 zop1 single mutant has decreased BDA1 transcript level As ZOP1 is a component of the splicing machinery (Zhang et al., 2013), the transcript level of many genes may be affected in the zop1 single mutant. To identify genes that are differentially expressed, RNA-seq was carried out on wild type and zop1-2 plants. Transcripts of 56 genes showed over 2-fold reduction and 23 genes showed over 2-fold increase in zop1-2 mutant compared to wild type plant. Gene ontology (GO) analysis of the biological functions of the differentially expressed genes revealed that genes related to defense responses are significantly enriched (Fig 3.6A). A large number of defense response-related genes were down-regulated in 61 the zop1-2 mutant, including BDA1, encoding a critical regulator of SNC2-mediated immunity (Figure 3.6B). To confirm the RNA-seq data, quantitative RT-PCR (RT-qPCR) analysis was performed. As shown in Figure 3.6C, transcript level of BDA1 was decreased in zop1-2 mutants. Previous RNA-seq analysis of the zop1 mutant in the C24 background revealed that a series of intron retention events occurred in the zop1 mutant (Du et al., 2015). Using rMATS approach, 377 intron-retention events in 350 genes were identified in zop1-2 mutant (adjust P value < 0.01). However, no intron retention event was detected in BDA1. PCR using primers flanking introns of BDA1 did not detect any alternatively spliced transcripts between wild type and zop1-2 (Figure 3.6D). Thus, ZOP1 seems only to affect the transcript level of BDA1 but not its pre-mRNA splicing process. 62 Figure 3.6 Analysis of defense gene expression in the zop1 mutants. (A) GO enrichment analysis of the biological functions of downregulated genes in zop1-2 compared to wild type. The top 10 significantly enriched GO terms ranked by enrichment scores are shown in bar graph. The plot graph indicates the number of downregulated genes in each GO term. 63 (B) Heatmap visualization of downregulated defense-related genes in zop1-2 compared to wild type. Each row of the heatmap represents a gene, each column represents a sample, and each cell displays normalized gene expression values. Blue color represents low expression and red color represents high expression. (C) Expression levels of BDA1 in the indicated genotypes as normalized by ACTIN1. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (D) Splicing pattern of BDA1 analysis on zop1-2 mutant. PCR was performed on cDNA or RNA samples under identical conditions. cDNA samples used in the PCR reactions were normalized using ACTIN1. 3.5 Discussion From a forward genetic screen that aims to identify suppressors of sard1-1 snc2-1D, we found that the pre-mRNA splicing factor ZOP1 is required for SNC2-mediated immunity. Mutations in ZOP1 partially suppresses the autoimmunity of sard1-1 snc2-1D, including the elevated PR gene expression, SA accumulation and enhanced disease resistance against a pathogen (Figure 3.1). Furthermore, zop1 single mutants exhibit compromised resistance against oomycete pathogen and reduced defense-related gene transcripts (Figure 3.5 and 3.6). Thus, our study suggests that, in addition to regulating pre-mRNA splicing, transcriptional gene silencing and cold stress responses, ZOP1 also has an important role in plant immunity regulation. ZOP1 has both RNA-directed DNA methylation (RdDM)-dependent and -independent roles in transcriptional silencing (Zhang et al., 2013). Here, we show that the effect of ZOP1 on the transcription level of BDA1 is not through the disruption of its pre-mRNA splicing. BDA1 executes a positive role in plant resistance against pathogens. Considering the requirement of ZOP1 in defense-related gene transcription and SNC2-mediated immunity, ZOP1 may regulate the BDA1 transcription through transcriptionally silencing the negative regulator(s) of BDA1 transcription and plant immunity. 64 GO term enrichment analysis on the zop1-2 mutant revealed that genes responsive to SA are differentially expressed in the zop1 mutant. Whether the regulation of ZOP1 is limited to SA-dependent immune responses needs further investigation. Moreover, among those differentially expressed genes in the zop1-2 mutant, some encode defense responses-related Cysteine-rich receptor-like kinases (CRKs). Arabidopsis CRKs form a subgroup of RLKs with more than 40 members (Chen, 2001). Their extracellular domains contain 2 copies of the conserved Cys-X8-Cys-X2-Cys motif. In the past decades, some CRKs were demonstrated to play critical roles in disease resistance in Arabidopsis. For example, overexpression of CRK4 and CRK36 leads to enhanced PTI responses (Yeh et al., 2015). As RLPs lack a cytoplasmic kinase domain, they were proposed to associate with RLKs to transduce defense signal to downstream components (Couto & Zipfel, 2016). Whether these CRKs participate in SNC2-mediated immunity would be interesting to examine in the future. 65 Chapter 4: The deubiquitinating enzyme AMSH1 contributes plant immunity through regulating the stability of BDA1 4.1 Summary Plants utilize plasma membrane localized receptor like kinases (RLKs) or receptor like protein (RLPs) to recognize pathogens and activate pattern-triggered immunity (PTI) responses. A gain-of-function mutation in the Arabidopsis RLP SNC2 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE 2) leads to constitutive activation of defense responses in snc2-1D mutant plants. Transcription factors, SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60g (CBP60g) define two parallel pathways downstream of SNC2. The autoimmunity of snc2-1D was partially affected by single mutations in SARD1 or CBP60g, but completely suppressed by the sard1 cbp60g double mutant. From a suppressor screen using sard1-1 snc2-1D, we identified a deubiquitinating enzyme ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM 1 (AMSH1) as a key component in SNC2-mediated plant immunity. Loss-of-function mutation in AMSH1 can suppress the autoimmune responses of sard1-1 snc2-1D. Selective protein degradation in eukaryotes often occurs through the ubiquitination/deubiquitination system. The deubiquitinating enzymes that remove ubiquitin from target proteins play essential roles in controlling the level of target protein ubiquitination and degradation. When AMSH1-like genes are silenced in Nicotiana (N.) benthamiana, decreased level of BDA1 was observed. As BDA1 is a transmembrane protein required for SNC2-mediated immunity, AMSH1 likely contributes to immunity regulation through controlling the stability of BDA1. 66 4.2 Introduction Plants have evolved effective defense mechanisms to thwart the surrounding microbial pathogens. Upon pathogen detection, the initial defense of plants begins with the recognition of evolutionarily conserved pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) at the cell surface, leading to pattern-triggered immunity (PTI) (Monaghan & Zipfel, 2012). Successful pathogens are able to suppress PTI through secreted effectors, which results in effector-triggered susceptibility (ETS) (Jones & Dangl, 2006). In turn, higher plants use intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) to recognize cognate effectors and turn on effector-triggered immunity (ETI) (Jones & Dangl, 2006). Activation of PTI or ETI results in a number of overlapping downstream outputs, such as activation of mitogen-activated protein kinase (MAPK) cascades, calcium flux, reactive oxygen species (ROS) burst, callose deposition, transcriptional reprograming and phytohormone signaling (Zhou & Zhang, 2020). PRRs are usually either receptor like kinases (RLKs) or receptor like proteins (RLPs) that lack an intercellular kinase domain (Boller & Felix, 2009). Arabidopsis SNC2 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE 2) encodes an RLP. Loss of SNC2 results in enhanced susceptibility to pathogenic bacteria Pseudomonas syringae pv tomato (Pst) DC3000, demonstrating that SNC2 is essential for basal resistance (Zhang, et al., 2010). The alteration of a G to R in a conserved motif of the transmembrane domain in snc2-1D results in smaller size and constitutively activated defense responses including elevated PATHOGENESIS-RELATED (PR) gene expression as well as defense hormone salicylic acid (SA) accumulation. Studies on snc2-1D-mediated autoimmunity revealed that BDA1 (for Bian Da; “becoming big” in Chinese), a novel ankyrin-repeat transmembrane protein, functions downstream of SNC2 to regulate plant immunity (Yang et al., 67 2012). Knocking out BDA1 leads to full suppression of snc2-1D autoimmunity, whereas a gain-of-function allele of BDA1, bda1-17D, exhibits constitutively activates cell death and defense responses. Two members of the Arabidopsis CALMODULIN-BINDING PROTEIN60 (CBP60) gene family, CBP60g and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1), encode positive regulators of plant immunity that promote the pathogen induced SA production (Wang et al., 2011; Zhang et al., 2010). Both genes are induced by virulent bacterial pathogen Pseudomonas syringae pv maculicola (Psm) ES4326 infection. Single mutants of either gene only exhibit modest increases in the growth of Psm ES4326. However, sard1 cbp60g double mutants allow greatly increased growth of Psm ES4326 and reduced levels of pathogen induced SA (Wang et al., 2009, 2011). Furthermore, Chromatin immunoprecipitation (ChIP)-seq analysis revealed that the expression of a battery of genes encoding key regulators of PTI and ETI is directly controlled by SARD1 and CBP60g during plant defense, demonstrating that SARD1 and CBP60g function as master regulators of plant immunity (Sun et al., 2015). Previous report showed that SARD1 and CBP60g contribute to autoimmunity of snc2-1D (Sun et al., 2015). The dwarf stature as well as constitutive activated immune responses are partially suppressed in sard1-1 snc2-1D and cbp60g-1 snc2-1D, and almost fully blocked in cbp60g-1 sard1-1 snc2-1D. These findings indicate that SARD1 and CBP60g define two parallel pathways downstream of SNC2 to mediate defense responses (Sun et al., 2015). However, how SNC2 activates SARD1 and CBP60g requires further investigation. 68 Plants use various ways to maintain protein homeostasis. One mechanism is through ubiquitin (Ub) mediated protein degradation. Ubiquitination is a reversible process, where the bond between Ub and the substrate can be cleaved by deubiquitinating enzymes (DUBs) to remove and rewrite the Ub modifications (Isono & Nagel, 2014). There are around 50 DUBs encoded in Arabidopsis genome, which can be classified into seven protein families according to the differences in their catalytic domains (Vogel & Isono, 2024). Different from the other six subfamilies belonging to cysteine proteases, the JAB1/MPN/MOV34 (JAMM) subfamily uses a zinc metalloproteinase domain for cleavage (Komander et al., 2009). RPN11, a DUB from the JAMM subfamily with an MPN+ domain (MPR1, PAD1 N-terminal+), has been implicated in hydrolyzing Ub chains prior to protein degradation by the 26S proteasome (Book et al., 2010). Additional MPN+ domain proteins, AMSH1 and AMSH3 were shown to be necessary for autophagic and endocytic degradation, respectively (Isono et al., 2010; Katsiarimpa et al., 2011, 2013, 2014). However, due to their broad target spectra, the specific substrates and physiological role of most DUBs are poorly defined. To identify regulators required for CBP60g-dependent defense responses downstream of SNC2, a suppressor screen was performed in the sard1-1 snc2-1D background. Here, we report the identification and characterization of suppressor 123-1 mutant. 123-1 carries a mutation in AT1G48790, which encodes a DUB ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM 1 (AMSH1). The DUB activity of AMSH1 toward Lys 63-linked Ub chains has been previously confirmed in vitro (Katsiarimpa et al., 2013). Our current analysis showed that AMSH1 is involved in regulating SNC2-mediated immune responses by affecting the protein level of BDA1. 69 4.3 Material and methods 4.3.1 Plant materials and growth conditions All Arabidopsis (A.) thaliana plants used in this study are in the Col-0 ecotype background. The sard1-1 snc2-1D mutant was previously described (Sun et al., 2015). amsh1-1 T-DNA insertion mutant SALK_108544C1 was obtained from Arabidopsis Biological Resource Center (ABRC). A CRISPR construct targeting AMSH1 was generated as previously described in chapter 2. For transgene complementation, the genomic DNA fragment of AMSH1 driven by its native promoter was PCR-amplified from wild-type genomic DNA and cloned into pBasta-3HA vector. Primers used for generating the constructs and genotyping the mutants are listed in Table 4.1. A. thaliana and Nicotiana (N.) benthamiana plants were grown on soil under long-day conditions (16-h light/ 8-h dark cycle) unless specified. Plants for pathogen-induced gene expression analysis and SA measurement were grown for two weeks under long-day conditions. The seedlings were then transplanted and grown for a further one or two weeks under short-day conditions (8-h light/16-h dark cycle) respectively. 4.3.2 Mutagenesis and suppressor screen Ethyl methane sulfonate (EMS) mutagenesis and screening M2 populations were carried out as described in chapters 2 and 3. 70 4.3.3 Mapping-by-sequencing 123-1 mutants were backcrossed with sard1-1 snc2-1D. In F2 population, 36 plants that showed similar morphology as original 123-1 mutant were selected and pooled for genomic DNA extraction. The genomic DNA was subsequently sequenced using the HiSeq-PE150 platform. Single nucleotide polymorphisms (SNPs) were then analyzed using a variant discovery pipeline based on Genome Analysis Toolkit (GATK) Best Practices run on the Compute Canada Cedar cluster (Huang et al., 2021). 4.3.4 Gene expression analysis Plants for quantitative reverse transcript-PCR (qRT-PCR) were grown on plates with ½ Murashige and Skoog (MS) and 1% sucrose for 12 days unless specified. To test Pst DC3000 hrcC- induced gene expression, leaves of 3-week-old plants grown under short-day conditions were syringe infiltrated with a bacterial suspension with OD600 of 0.05 in 10 mM MgCl2 and collected at 0 h and 12 h post inoculation. RNA extraction, reverse transcription and qPCR were carried out as described in chapters 2 and 3. 4.3.5 Infection assay Hyaloperonospora arabidopsidis (Hpa) Noco2 infection assays were performed as described in chapters 2 and 3. 4.3.6 SA measurement For pathogen-induced SA accumulation analysis, 4-week-old plants grown under short-day conditions were infiltrated with Pst DC3000 hrcC- at a dose of OD600=0.05. Samples were 71 harvested at 0 h and 12 h after infiltration. SA was extracted and measured with high-performance liquid chromatography (HPLC) as described in chapters 2 and 3. 4.3.7 RNAi construct design and transient expression in N. benthamiana The RNAi constructs were made using an intron‐based method for dsRNA generation (Varsha Wesley et al., 2001). Using WebApollo LAB3.6 genome browser platform, a gene with two copies, NbL15g10460.1 and NbL03g21980.1, was identified as an AMSH1-like gene in N. benthamiana (referred to NbAMSH1 in this study) (Ranawaka et al., 2023). The 300 bp target sequence for silencing NbAMSH1 was selected by SGN VIGS Tool (Zulfiqar et al., 2023). The sense and antisense fragments of the target were PCR-amplified from wild type N. benthamiana genomic DNA. Intron 3 of PYRUVATE ORTHOPHOSPHATE DIKINASE (PDK) from Flaveria trinervia was used as a spacer sequence between sense and antisense gene fragments to generate a harpin structure. Then sense fragment, PDK intron and antisense fragment were accordingly assembled into pCambia1300-35S-E9ter using NEBuilder® HiFi DNA Assembly Kit (NEW ENGLAND Biolabs, Canada). The primers used for making RNAi construct were listed in Table 4.1. For transient expression in N. benthamiana, empty vector pCambia1300 (OD600=0.4) and BDA1-HATurboID (OD600=0.2) were infiltrated into the left halves of four‐week‐old N. benthamiana leaves as control. The RNAi constructs carrying fused sense‐intron‐antisense fragments of NbAMSH1 (OD600=0.4) and BDA1-HATurboID were expressed in the right half leaves. After infiltration, the plants were kept in dark for 2 days to induce the expression of RNAi constructs. Total protein was extracted from the infiltrated area at 48 hours post infiltration and then subjected to immunoblot analysis. 72 Table 4.1 Primers used in Chapter 4. Primer name Primer sequence (5’ to 3’) AMSH1-T-DNA-F TATGGTTTATCCAACCATGGG AMSH1-T-DNA-R TAGTTTCACTTTCAAACGCGC AMSH1-pro-EcoRI-F GACTGAATTCCTGACAACAGTTACAGGCATCC AMSH1-StuI-R GAGAAGGCCTTCTGAGATCAATGACATCAA Nb-AMSH1-F1 ATTTGGAGAGAACAGAATTCGAGCTCTCCTAAAGTTGAAAATTTG Nb-AMSH1-R1 TTATTTCCTTACCAATTGGGGTACCCTTGGGACTTGAGACACAAA Nb-AMSH1-F2 GTTCGAAATCGATAAGCTTGGATCCCTTGGGACTTGAGACACAAA Nb-AMSH1-R2 TAAGCTTGCATGCCTGCAGTCTAGATCCTAAAGTTGAAAATTTGT 4.4 Results 4.4.1 Identification and characterization of suppressor mutant 123-1 An EMS forward genetic screen was conducted in sard1-1 snc2-1D to identify regulators required for CBP60g-dependent defense responses downstream of SNC2. As shown in Figure 4.1A, the 123-1 mutants displayed intermediate plant size compared to wild type Col-0 and sard1-1 snc2-1D. qRT-PCR analysis showed that the constitutive expression of PR1 and PR2 in sard1-1 snc2-1D was largely suppressed in 123-1 (Figure 4.1B and 4.1C). In addition, SA level in the 123-1 mutants was greatly reduced compared with sard1-1 snc2-1D (Figure 4.1D). Moreover, the enhanced disease resistance against oomycete pathogen Hpa Noco2 in sard1-1 snc2-1D was severely compromised in 123-1 mutant (Figure 4.1E). 73 Figure 4.1 Identification and characterization of the sard1 snc2-1D suppressor mutant 123-1. (A) Morphology of three-week-old soil-grown plants of the indicated genotypes under long-day condition. Scale bar is 1 cm. (B and C) Relative expression levels of PR1 (B) and PR2 (C) in the indicated genotypes. Transcript levels were normalized with ACTIN1. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (D) Free SA levels in the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (E) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). 4.4.2 Sequencing-by-mapping of 123-1 To identify the causal mutation in 123-1, whole genome sequencing (WGS) was performed on a 123-1 mapping population. When 123-1 was backcrossed to sard1-1 snc2-1D, the resulting F1 plants displayed sard1-1 snc2-1D-like morphology (Figure 4.2A). Further, in the F2 progeny, a 48:136 segregation of suppressor-like and sard1-1 snc2-1D-like plants was observed, which 74 matches to the expected 1:3 ratio (χ2 = 0.1127; P value = 0.7375), suggesting that the autoimmune suppressing phenotypes of 123-1 are due to a single recessive nuclear mutation. F3 seeds from the suppressor-like F2 plants were harvested and planted. Tissue was collected for DNA extraction from 34 confirmed homozygous F3 populations. Following the WGS and data analysis, a genetic linkage region was identified on Chromosome 1 (Figure 4.2B). In this region, one of the candidate genes, At1g48790 (encoding AMSH1), contains a G to A mutation at the splice junction of the tenth exon. Figure 4.2 Mapping-by-sequencing of 123-1. (A) Morphology of 24-day-old soil-grown plants of the indicated genotypes under long-day condition. Scale bar is 1 cm. (B) Linkage region for the 123-1 mutation in the middle of chromosome 1. The mutation in AMSH1 is highlighted using a red data point. 75 4.4.3 Loss of AMSH1 suppresses the autoimmunity of sard1-1 snc2-1D To determine whether the mutant phenotype of 123-1 is caused by the mutation in AMSH1, a CRISPR/Cas9 construct with two gRNAs targeting AMSH1 was generated and transformed into the sard1-1 snc2-1D plants. Two independent CRISPR lines were recovered with deletions inside the gene (Figure 4.3A). Similar to the original suppressor mutant 123-1, these deletion lines displayed a larger size than sard1-1 snc2-1D (Figure 4.3B). The constitutive expression of PR1 and PR2 was suppressed in these CRISPR lines as well (Figure 4.3C and 4.3D). Consistently, the elevated SA accumulation as well as enhanced disease resistance against Hpa Noco2 was blocked in these deletion lines (Figure 4.3E and 4.3F). Lastly, the wild type AMSH1 under its own promoter was transformed into the 123-1 mutant. As shown in Figure 4.4A and Figure 4.4B, AMSH1 complemented the 123-1 phenotypes (Figure 4.3F). The CRISPR and complementation data suggest that the amsh1 mutation in 123-1 is the causal one for the suppressor, and AMSH1 is required for the autoimmunity of sard1-1 snc2-1D. 76 Figure 4.3 Deletion mutations of AMSH1 generated through CRISPR suppress the autoimmunity of of sard1-1 snc2-1D. (A) Mutations in At1g48790. 123-1 carries a G to A substitution at the splice junction of the tenth exon in At1g48790 (top). #1 and #2 are two independent CRISPR deletion lines of AMSH1 in the sard1-1 snc2-1D background, deleting the same genomic region (bottom). (B) Morphology of 24-day-old soil-grown plants of the indicated genotypes under long-day condition. Scale bar is 1 cm. (C and D) Expression levels of PR1 (C) and PR2 (D) in the indicated genotypes as normalized by ACTIN1. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (E) Free SA level in the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). (F) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). 77 Figure 4.4 pAMSH1::AMSH1 complements the phenotypes of the suppressor 123-1. (A) Morphology of 21-day-old soil-grown plants of the indicated genotypes under long-day condition. #5 and #6 are two independent complementation lines of AMSH1 in the 123-1 background. Scale bar is 1 cm. (B) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Error bars represent standard deviations. Letters indicate statistical differences (P < 0.05, one-way ANOVA; n = 3). 4.4.4 amsh1-1 single mutant exhibits enhanced basal immunity and PTI responses To dissect the role of AMSH1 in plant immunity, infection experiments were performed using the amsh1 T-DNA insertion allele in Col-0 background (referred to as amsh1-1 in this study). Firstly, the obligate biotrophic oomycete pathogen Hpa Noco2 was used to inoculate wild type and amsh1-1 plants. As shown in Figure 4.5A, amsh1-1 mutant supported significantly less Hpa Noco2 growth than wild type plants. This is consistent with a previous report that the amsh1 T-DNA insertion allele CSHL_ET8678 in Ler background exhibited enhanced resistance against obligate biotroph Erysiphe cruciferarum (Katsiarimpa et al., 2013). In addition, higher PR1 accumulation was 78 detected in the amsh1-1 mutant, suggesting that amsh1-1 mutant exhibit enhanced basal immunity (Figure 4.5B). Next, amsh1-1 plants were challenged with the type III secretion system deficient pathogen Pst DC3000 hrcC-, which triggers PTI. Consistent with the enhanced basal immunity, the amsh1-1 mutant displayed higher level of CBP60g expression compared to wild type plants under mock treatment. Upon hrcC- infiltration, amsh1-1 plants also showed higher expression level of CBP60g than wild type (Figure 4.5C). The difference of basal SA levels in wild type and amsh1-1 mutant was barely detectable with mock treatment, while the SA level was obviously higher in amsh1-1 mutant than in wild type with Pst DC3000 hrcC- infection (Figure 4.5D), suggesting that amsh1-1 also exhibits enhanced PTI responses. Figure 4.5 Enhanced basal immunity and PTI in the amsh1-1 single mutant. 79 (A) Growth of Hpa Noco2 conidiospores on the indicated genotypes. Error bars represent standard deviations. **** indicates statistical differences (P < 0.05, unpaired t test; n = 4). (B) Expression level of PR1 in the indicated genotypes as normalized by ACTIN1. RNA was extracted from 25-day-old soil-grown plants. Error bars represent standard deviations. * indicates statistical differences (P < 0.05, unpaired t test; n = 3). (C) Relative expression levels of CBP60g in the indicated genotypes treated with 10 mM MgCl2 (mock) or Pst DC3000 hrcC- (OD600 = 0.05) for 12 h. Values were normalized to the expression of ACTIN1. Error bars represent standard deviations. * indicates statistical differences (P < 0.05, unpaired t test; n = 3). (D) SA levels in the indicated genotypes treated with 10 mM MgCl2 (mock) or Pst DC3000 hrcC- (OD600 = 0.05) for 12 h. Error bars represent standard deviations. ** indicates statistical differences (P < 0.05, unpaired t test; n = 4). 4.4.5 Silencing AMSH1-like genes in N. benthamiana negatively affects BDA1 protein levels AMSH1 is an active DUB. Its catalytic MPN+ domain specifically cleaves Lys 63-linked polyubiquitin chains from substrates to release both the Ub and the target, preventing the degradation of the pre-targeted protein (Komander et al., 2009). Considering the requirement for AMSH1 in the constitutive defense responses of sard1-1 snc2-1D, we hypothesized that AMSH1 may prevent the degradation of key regulators in SNC2-mediated immunity, such as BDA1 (Yang et al., 2012). To test this, we silenced the homologous genes of AMSH1 in N. benthamiana and examined whether the protein level of BDA1 was affected. As shown in Figure 4.6A and B, comparing with the control group, co-infiltration of RNAi construct carrying the NbAMSH1 fragment with the construct expressing BDA1-HATurboID resulted in decreased BDA1 protein levels in N. benthamiana leaves, suggesting that AMSH1 exerts a role in preventing BDA1 degradation. 80 Figure 4.6 Silencing AMSH1-like genes in N. benthamiana leads to decreased protein levels of BDA1. (A) Immunoblot analysis of protein levels of BDA1-HA-TurboID in the absence or presence of RNAi NbAMSH1 construct in N. benthamiana. Agrobacterium carrying plasmids expressing BDA1-HA-TurboID together with EV-3FLAG or RNAi NbAMSH1 were co-infiltrated into one half of each N. benthamiana leaf. Samples were collected at 48 h post-inoculation (hpi). Equal loading is shown by Ponceau S staining of a non-specific band. Protein abundance was quantified using ImageJ (https://imagej.nih.gov/ij/). (B) Quantification of relative band intensity of (A). The intensity of BDA1-HATurboID bands upon co-infiltration with EV-FLAG was set as 1. Error bars represent standard deviations. **** indicates statistical differences (P < 0.05, unpaired t test; n = 6). 4.5 Discussion From the suppressor screen of sard1-1 snc2-1D to identify regulators involved in CBP60g-dependent resistance pathways downstream of SNC2, we found that the DUB AMSH1 contributes to snc2-1D autoimmunity. Mutation in AMSH1 leads to suppression of the autoimmune responses in sard1-1 snc2-1D plants, including the elevated PR gene expression, SA accumulation and enhanced disease resistance (Figure 4.1). The constitutive defense responses of snc2-1D specifically requires BDA1, a transmembrane protein with unknown function (Yang et al., 2012). Silencing AMSH1 homologs in N. benthamiana results in less BDA1 protein levels, suggesting that AMSH1 may contribute to SNC2-mediated immunity by affecting BDA1 protein abundance. Whether BDA1 is a direct substrate of AMSH1 requires further investigation. 81 Surprisingly, amsh1 single mutant exhibits enhanced basal resistance and PTI responses, with higher PR gene transcripts as well as SA levels (Figure 4). This is mostly likely due to the role of AMSH1 in regulating intracellular trafficking and degradation of endocytic cargoes other than BDA1 (Vogel & Isono, 2024). The complex phenotype of amsh1 could represent the combined effects from different substrates of AMSH1. Studies have shown that ubiquitination/deubiquitination processes contribute to the regulation of endocytosis and the subsequent vacuolar degradation of plasma membrane receptors in plants, including REQUIRES HIGH BORON 1 (BOR1), FLAGELLIN-SENSITIVE 2 (FLS2) and PIN-FORMED 2 (PIN2) (Abas et al., 2006; Göhre et al., 2008; Kasai et al., 2011). amsh1 mutants are defective in Endosomal Sorting Complexes Required for Transport (ESCRT)-mediated vacuolar targeting (Katsiarimpa et al., 2013). Specifically, enzymatic inactive AMSH1 inhibits AvrPtoB-dependent endocytic degradation of CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) (Katsiarimpa et al., 2014). It remains to be determined whether AMSH1 contributes to the endocytic degradation of SNC2 or even its paired unknown RLK or BDA1. The Arabidopsis genome encodes three AMSH family members, namely AMSH1, AMSH2 and AMSH3. The amino acid sequences of AMSH1 and AMSH3 are more similar to each other, than they are to AMSH2, sharing 47% overall amino acid identity (Katsiarimpa et al., 2011, 2013). They possess an N-terminal microtubule interacting and transport (MIT) domain, and a C-terminal MPN+ domain (Isono et al., 2010). Both AMSH1 and AMSH3 interact with endocytosis machinery through the MIT domain (Katsiarimpa et al., 2011, 2013). Their characteristic MPN+ domain have two zinc ions. One of the two zinc ions activates water molecules to promote the 82 cleavage of isopeptide bonds on Lys 63-linked polyubiquitin chains (Sato et al., 2008). Thus, AMSH1 and AMSH3 share similar biochemical properties. AMSH3 was reported to be required for the ETI responses mediated by coiled-coil type nucleotide-binding leucine-rich repeat receptors (CC-NLRs, CNLs) RPM1 (RESISTANCE TO P. SYRINGAE PV MACULICOLA 1) and RPS2 (RESISTANT TO P. SYRINGAE 2) (Schultz-Larsen et al., 2018). Whether AMSH1 also contributes to CNL-mediated resistance remains unclear. Overall, the requirement of DUB AMSH1 for SNC2-mediated immunity revealed another layer of regulation of SNC2-mediated immunity. And the characterization of the amsh1 mutant uncovered a crucial role of AMSH1 in PTI responses. 83 Chapter 5: Conclusions and future directions 5.1 Conclusions Arabidopsis SNC2 encodes an RLP with a pivotal role in plant basal resistance and PTI responses (Zhang et al., 2010). A gain-of-function allele of SNC2, snc2-1D, exhibits autoimmunity, including enhanced PR gene expression and elevated levels of SA in the absence of pathogens, leading to its small size with dark green and curly leaves. The distinctive phenotypes of snc2-1D provide a unique background to perform robust genetic analyses to study SNC2-mediated immunity in Arabidopsis. By screening the suppressor mutants of snc2-1D, several regulators of SNC2-mediated immunity have been identified. As an example, BDA1 is a novel ankyrin-repeat transmembrane protein that functions as a positive regulator of plant resistance (Yang et al., 2012). The CaM-binding transcription factor CBP60g function in parallel with its homolog SARD1 to regulate immune responses downstream of SNC2; loss of either CBP60g or SARD1 alone partially suppresses the autoimmunity of snc2-1D (Sun et al., 2015). However, how the plasma membrane localized SNC2 transduces signals to CBP60g is largely unknown. My forward genetic screen was carried out in the Arabidopsis sard1-1 snc2-1D background to identify novel components contributing to CBP60g-dependent signaling pathway downstream of SNC2. In this thesis, I summarize the identification and characterization of three isolated suppressor mutants, and how these proteins regulate plants defense responses downstream of SNC2. Chapter 2 describes the identification and characterization of a full suppressor of sard1-1 snc2-1D, 125-1. Mapping-by-sequencing and genetic analysis revealed that 125-1 carries a gain-of-function mutation in ECA4, which encodes an epsin-like clathrin adaptor. Further analysis found that ECA4 84 is a negative regulator of plant immunity and most likely executes its function by modulating the turnover of BDA1. Chapter 3 describes a partial suppressor of sard1-1 snc2-1D, 116-1. Through mapping-by-sequencing, a premature stop codon in ZOP1 was identified, causing the partial suppression of sard1-1 snc2-1D. ZOP1 encodes a pre-mRNA splicing factor involved in promoting RdDM and transcriptional silencing. RNA-seq analysis with zop1-2 revealed that several defense responsive genes were downregulated in zop1-2 single mutant, including BDA1. Thus, ZOP1 positively contributes to plant immunity through regulating the transcript levels of defense related genes. Chapter 4 uncovers the role of DUB AMSH1 in SNC2-mediated immunity. Loss-of-function mutation in AMSH1 caused partial suppression of sard1-1 snc2-1D’s autoimmunity. As silencing AMSH1 homologs in N. benthamiana results in less BDA1 protein levels, AMSH1 likely regulates BDA1 protein stability. Interestingly, amsh1 single mutant exhibits enhanced basal resistance and PTI responses, which is likely the combined effects from different substrates of AMSH1. Overall, my PhD thesis work provides new insights into how SNC2-mediated signaling pathways are regulated. The requirement of ECA4, ZOP1 and AMSH1 for SNC2-mediated immunity revealed other layers of regulation of SNC2-mediated immunity, which are clathrin-mediated endocytosis, pre-mRNA splicing and ubiquitination/deubiquitination processes respectively. My studies offered evidence that the clathrin adaptor ECA4 and DUB AMSH1 differentially regulate the protein level of BDA1, whereas the splicing factor ZOP1 regulates the transcript level of BDA1. These findings also highlight the significant role of BDA1 in SNC2-mediated defense signaling 85 pathway, that the SNC2-mediate immune outputs were mainly optimized through the regulation on BDA1 homeostasis. Figure 5.1 A work model of SNC2-mediated signaling pathway. BDA1 serves as a hub in SNC2-mediated signaling pathway. The clathrin adaptor ECA4, DUB AMSH1 and splicing factor ZOP1 contribute to SNC2-mediated immunity by differentially modulating the homeostasis of BDA1. 5.2 Future directions While studies on the mutants of ECA4, ZOP1 and AMSH1 improved our understanding of the regulation of the defense signaling downstream of SNC2, many questions remain to be addressed. 5.2.1 Function of ECA4 in plant immunity ECA4 and CAP1 are two homologous ANTH-type clathrin adaptors with high sequence identity. Results in chapter 2 revealed their redundant functions in regulating plant immunity, eca4 or cap1 86 single mutant does not show altered plant defense responses whereas eca4 cap1 double mutant are defective in resistance against oomycete pathogen. Another research group reported their possible redundant contribution to the CME process in sensitized endocytic mutant background (Adamowski et al., 2022). However, unlike overexpression of ECA4, which results in suppression of the sard1-1 snc2-1D phenotypes, transgenic lines overexpressing CAP1 in sard1-1 snc2-1D are still sard1-1 snc2-1D like (data not shown), suggesting that ECA4 may play a more dominant role in regulating plant immunity comparing with CAP1. In addition, the E357 residue of ECA4 is conserved in CAP1. Whether the corresponding E to K mutation in CAP1 would result in any interference in its function and the sard1-1 snc2-1D’s autoimmunity would be interesting to test. Previous studies showed that a GFP fused ECA4 protein primarily localized to the TGN/EE and, to a lesser extent, to the plasma membrane (Nguyen et al., 2018). Although overexpression of GFP tagged ECA4 does not affect its localization (Nguyen et al., 2018), we cannot rule out the possibility that the E357K mutation in ECA4 changes its subcellular distribution preference. Our result indicates that ECA4 negatively regulates plant immunity through modulating the transmembrane protein BDA1 levels. Such modulation could be direct or indirect. ECA4 may interact with the transmembrane domain of BDA1, and the subcellular distribution change caused by eca4-4D may affect their interaction or change the subcellular localization of BDA1 for the subsequent CME. To test this hypothesis, observing the subcellular distribution of ECA4E357K-GFP and BDA1-GFP, and testing the interaction between ECA4 and BDA1 would be necessary. Since ECA4 may contribute to the CME of plasma membrane proteins, we also tested whether eca4-4D mutation affects the RLP SNC2 protein levels by crossing eca4-4D single mutant with 87 HA tagged SNC2 transgenic lines. As a result, HA tagged SNC2 transgenic plants carrying homozygous eca4-4D mutation did not show reduced protein abundance comparing with their parent HA tagged SNC2 transgenic plant (data not shown). Thus, ECA4 may not participate in the internalization of protein SNC2. Alongside the TPLATE complex, the heterotetrameric adaptor protein complex AP2 also facilitates the CME of various plasma membrane localized proteins (Gadeyne et al., 2014). The unstructured region of ECA4 contains a single DPF motif involved in weak molecular interactions with the AP2 complex (Smith et al., 2017), which is composed of four subunits, AP2ε, AP2β, AP2μ and AP2σ (Yamaoka et al., 2013). We found that knocking out the AP2σ subunit (AP2S; At1g47830) or the AP2μ subunit (AP2M; At5g46630) result in partial suppression of the dwarfism of snc2-1D npr1-1 eds5-3 (data not shown), suggesting that AP2 complex also contributes to SNC2-mediated immunity. Further characterization on AP2S and AP2M deletion alleles would provide more information on the CME-mediated regulation of SNC2-mediated immune responses. 5.2.2 Function of ZOP1 in plant immunity Studies in chapter 3 indicated that ZOP1 is involved in SNC2-mediated immunity. ZOP1 is a pre-mRNA splicing factor that associates with components of the splicing machinery including STA1 and RDM16 (Du et al., 2015). Previous RNA deep sequencing on zop1 mutant in the C24 background revealed its critical role in plant tolerance to chilling stress (Du et al., 2015). Our RNA-seq analysis on zop1-2 mutant in the Col-0 background shows that, in addition to BDA1, the transcript of RLP23 was also significantly lower (Figure 3.6B). RLP23 senses the PAMP nlp20 and forms a heterotrimer with BAK1 and SOBIR1 to activate the downstream defense responses 88 (Albert et al., 2015). In the future, examining the nlp20-induced responses in zop1 mutant would help to better understand the role of ZOP1 in PTI. Loss of ZOP1 may cause defects in pre-mRNA splicing (Zhang et al., 2013), therefore analysis of the genome-wide alternative splicing events in zop1 mutant to would be required to examine whether ZOP1 also affects the alternative splicing of genes contribute to PTI and ETI. 5.2.3 Function of AMSH1 in plant immunity Data in chapter 4 showed that amsh1 mutation accounts for the suppression of snc2-1D-mediated autoimmunity, while amsh1 single mutant exhibits enhanced basal resistance and PTI. Such a complex phenotype may be due to the combined effects from different substrates of AMSH1, which is a DUB. Silencing AMSH1 homologs in N. benthamiana results in less BDA1 protein levels. Whether BDA1 is a direct target of AMSH1 can be answered by testing the interaction between AMSH1 and BDA1. Previously, Katsiarimpa et al. proposed two possibilities for AMSH1’s function in plant immunity, either SA-dependent basal immunity and cell death are indirectly affected in the Ler amsh1 mutant or AMSH1 directly regulate plant defense (Katsiarimpa et al., 2013). If BDA1 is a substrate of AMSH1, it would provide good support for the second hypothesis. 5.2.4 Function of BDA1 in plant immunity Studies in chapter 2,3 and 4 revealed that ECA4, ZOP1 and AMSH1 converge on BDA1 to regulate SNC2-mediated immunity. However, the biochemical function of BDA1 as well as the mechanism of how BDA1 regulates plant immunity is still enigmatic. Apart from BDA1, Arabidopsis genome encodes another 36 predicted ankyrin-repeat transmembrane proteins, such as ITN1 and ACD6, 89 participating in various physiological and developmental processes, including immunity (Becerra et al., 2004). Arabidopsis INCREASED TOLERANCE TO NACL 1 (ITN1) participates in salt-stress tolerance through its effect on abscisic acid (ABA)-induced production of ROS under salt stress (Sakamoto et al., 2008). ACCELERATED CELL DEATH 6 (ACD6) was found to associate with different PRRs in planta to in response to different PAMPs (Tateda et al., 2014; Zhang et al., 2014). Recently, ACD6 was proposed to function as an ion channel associated with calcium influx in plant immunity (Chen et al., 2023). It requires further investigation whether BDA1 is a Ca2+-conducting channel, like ACD6, that contributes to the regulation of calcium influx, therefore activates the downstream CaM binding transcription factor CBP60g for defense responses. 5.2.5 Identification of signaling partner(s) of SNC2 in plant immunity As RLPs lack intracellular kinase region, they often associate with RLKs to transduce defense signals (Albert et al., 2015; Couto & Zipfel, 2016; Liebrand et al., 2014). During the past years, several independent suppressor screens have been performed on snc2-1D-containing background (snc2-1D npr1-1, snc2-1D npr1-1 eds5-3 and cbp60g snc2-1D) to search for the missing RLKs functioning with SNC2 (Ding et al., 2018; Huang et al., 2021; Yang et al., 2012; Zhang et al., 2010). However, none of them successfully identified the partner RLKs of SNC2. This might be due to genetic redundancy. Alternatively, the co-receptors of SNC2 may be essential for plant development. Another plausible explanation could be that the SNC2 co-receptors are guarded by NLR proteins. Mutations in the co-receptors suppress the autoimmunity of scn2-1D, meanwhile, the NLR-mediated immunity is also hyperactivated and still causes autoimmunity-related small stature. In the future, biochemical approaches can be utilized to look for potential interactors of SNC2. 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Thesis/Dissertation
eng
Botany
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Zhang, Yuelin
Li, Xin
Genetic analysis of defense pathway downstream of the immune receptor SNC2 in Arabidopsis thaliana
Text