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Genetic analysis of anp2/3-mkk6-mpk4 cascade in plant immunity Lian, Kehui 2016

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   Genetic Analysis of ANP2/3-MKK6-MPK4 Cascade in Plant Immunity   by   Kehui Lian   B.Sc., Nankai University, 2013   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF    MASTER OF SCIENCE  in   The Faculty of Graduate and Postdoctoral Studies  (Botany)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)    April 2016   © Kehui Lian, 2016  ii   Abstract Recognition of pathogens through pathogen-associated molecular patterns (PAMPs) or effectors in plants activates a variety of defense responses including MAPKs signaling pathways and defense related genes expression. ANPs (Arabidopsis Nucleus- and Phragmoplast- localized kinase 1 related protein kinases), including ANP1, ANP2, ANP3 are three MAP kinase kinase kinases that form a MAP kinase cascade with downstream MKK6 and MPK4 to regulate cytokinesis process. In this study, we showed that the anp2 anp3 double mutants exhibit constitutive expression of PR (Pathogenesis-Related) genes and enhanced resistance against oomycete pathogen H. a. Noco2, suggesting that ANP2 and ANP3 negatively regulate plant immunity. In addition, loss function of MKK6 causes high levels of PR gene expression, indicating that MKK6 is involved in negative regulation of defense responses. Since MPK4 was previously shown to function as a negative regulator of plant immunity, we tested whether MPK4 functions downstream of ANP2/ANP3 and MKK6 in plant immunity by introducing CA-MPK4 transgene, which expresses a constitutively active (CA) variant of MPK4, to anp2 anp3 and mkk6. Constitutive expression of PR genes and enhanced resistance to H.a. Noco2 in anp2 anp3 and mkk6 were partially suppressed by expressing CA MPK4, suggesting that the ANP2/ANP3, MKK6 and MPK4 function in a MAPK cascade to negatively regulate defense responses.  To find out components that function downstream of ANP2/ANP3- MKK6-MPK4 cascade in plant immunity, two mutants summ2-8 (SUPPRESSOR OF MKK1 MKK2 2) and pad4-1 (PHYTOALEXIN DEFICIENT 4) were crossed into anp2 anp3 respectively. The constitutive defense responses in anp2 anp3 were fully suppressed by pad4-1, but not affected by the summ2-8 mutation,  iii  suggesting that PAD4 functions downstream of ANP2/ANP3 and that immune responses mediated by certain TIR-NB-LRR R proteins might be activated in the anp2 anp3 mutant.     iv  Preface   The work described in this thesis is the culmination of research from September 2013 through March 2016. Below is the manuscript (in preparation) that comprises this thesis and the contribution made by the candidate.   Chapter 2 – Arabidopsis MKK6 functions in two parallel MAP kinase cascades that negatively regulate plant immunity was modified from a prepared manuscript: The work is done in collaboration with Fang Gao. The candidate performed the following experiments under the instruction of Yuelin Zhang: PR gene expression in mkk6 and CA-MPK4 mkk6; characterization of anp2 anp3, anp2 anp3 CA-MPK4, anp2 anp3 pad4-1 and anp2 anp3 summ2-8; Pto DC3000 hrcC- infection assay. Fang Gao conducted the following experiments: characterization of summ4-1D mkk1 mkk2; positional cloning of SUMM4; PR gene expression in summ4-1D,  mekk1, mpk4, summ4-1D mekk1 and summ4-1D mpk4 ; interaction of MKK6 with MEKK1 and MPK4. Yuelin Zhang, Fang Gao and the candidate prepared the manuscript.    v  Table of Contents Abstract ............................................................................ ii Preface ............................................................................ iv Table of Contents ............................................................ v List of Figures .............................................................. viii List of Abbreviations...................................................... ix Acknowledgements ...................................................... xii Chapter 1 Introduction .................................................... 1 1.1 Plant immunity ................................................................................... 1 1.2 PAMP-triggered immunity ................................................................. 1 1.2.1 PRRs and PAMPs ......................................................................2 1.2.1.1 FLS2 and flg22 .................................................................. 2 1.2.1.2 EFR and EF-Tu .................................................................. 2 1.2.1.3 CERK1 and chitin ............................................................. 3 1.2.2 Downstream defense responses .............................................3 1.2.2.1 Callose deposition ............................................................ 3 1.2.2.2 ROS production ................................................................ 4 1.2.2.3 MAPK cascade activation ................................................ 4 1.2.3 Signaling components downstream of PAMP receptors .......6 1.3 Effector-triggered susceptibility ....................................................... 7 1.4 Effector-triggered immunity.............................................................. 8 1.4.1 R protein signaling ...................................................................9 1.5 Roles of ANPs in plant development and immunity ....................... 9 1.5.1 ANPs regulate cytokinesis process ........................................9 1.5.2 Roles of ANPs in immune responses .................................... 10  vi  1.5.3 MKK6 and MPK4 function downstream of ANPs to regulate cytokinesis ....................................................................................... 11 Chapter 2 Arabidopsis MKK6 functions in two parallel MAP kinase cascades that negatively regulate plant immunity ........................................................................ 13 2.1 Summary .......................................................................................... 13 2.2 Introduction ...................................................................................... 14 2.3 Results .............................................................................................. 16 2.3.1 Characterization of summ4-1D mkk1 mkk2 .......................... 16 2.3.2 Positional cloning of SUMM4 ................................................. 18 2.3.3 The summ4-1D mutation results in elevated expression of MKK6 ................................................................................................. 21 2.3.4 summ4-1D does not suppress the autoimmune phenotypes of mekk1 and mpk4.......................................................................... 21 2.3.5 MKK6 interacts with MEKK1 and MPK4 ................................ 24 2.3.6 PR1 and PR2 are constitutively expressed in mkk6 mutant plants ................................................................................................ 26 2.3.7 Defense responses are constitutively activated in the anp2 anp3 double mutant ......................................................................... 28 2.3.8 The autoimmune phenotype of anp2 anp3 can be partially suppressed by the CA-MPK4 mutant ............................................. 30 2.3.9 Constitutive defense responses in anp2 anp3 are independent of SUMM2 ................................................................... 32 2.3.10 PAD4 is partially required for the autoimmune phenotype of anp2 anp3 ......................................................................................... 32 2.3.11 anp2-2 anp3-3 is more susceptible to P. syringae pv tomato DC3000 hrcC- .................................................................................... 35 2.4 Discussion ....................................................................................... 36 2.5 Methods ............................................................................................ 39 2.5.1 Plant materials ........................................................................ 39 2.5.2 Mutant characterization .......................................................... 39 2.5.3 Map-based cloning of SUMM4 ............................................... 40 2.5.4 Split luciferase assay ............................................................. 41 Chapter 3 Future directions and conclusions ............ 42  vii  References ..................................................................... 43     viii   List of Figures  Figure 1 Characterization of summ4-1D mkk1 mkk2. .................................... 17 Figure 2 Positional cloning of SUMM4. ........................................................... 20 Figure 3 summ4-1D does not suppress the autoimmune phenotypes of mekk1 and mpk4. .............................................................................................. 23 Figure 4 Interactions between MKK6 and MEKK1 or MPK4. ......................... 25 Figure 5 Expression of PR genes in mkk6 and CA-MPK4 mkk6. .................. 27 Figure 6 Characterization of the anp2 anp3 double mutant. ......................... 29 Figure 7 CA-MPK4 partially blocks the constitutive defense responses in anp2-2 anp3-3. ................................................................................................... 31 Figure 8 Constitutive defense responses in anp2-2 anp3-3 are independent of SUMM2 but dependent on PAD4. ................................................................ 34 Figure 9 Growth of Pto DC3000 hrcC- in wild type, anp2-2, anp3-3, and anp2-2 anp3-3. ................................................................................................... 35 Figure 10 A working model for the roles of MKK6 in plant immunity. .......... 38      ix  List of Abbreviations ANPs                   Arabidopsis Nucleus- and Phragmoplast-                         localized kinase 1 related protein kinases ASR3                   Arabidopsis SH4-related 3 AvrB      an avirulence protein from Pseudomonas syringae AvrRpm1     an avirulence protein from Pseudomonas syringae AvrRpt2                 an avirulence protein from Pseudomonas syringae AvrPto                  an avirulence protein from Pseudomonas syringae AvrPtoB     an avirulence protein from Pseudomonas syringae BAK1      BRI1-Associated Kinase 1 BiFC      Biomolecular Fluorescence Complementation  BIK1      Botrytis- Induced Kinase 1 BRI1      Brassinosteroid- Insensitive 1 CA       Constitutively active CaMV 35S promoter a very strong constitutive promoter found in                          Cauliflower mosaic virus CC       Coiled-coil CERK1      Chitin Elicitor Receptor Kinase 1 CFU      Colony-Forming Units Co-IP      Co-Immunoprecipitation Col-0  an Arabidopsis ecotype; it’s referred as wild type in this thesis DAMP      Danger- Associated Molecular Pattern DNA      Deoxyribonucleic acid E.coli      Escherichia coli EDS1      Enhanced Disease Susceptibility 1 EFR      EF-Tu Receptor  x  EF-Tu      bacterial translation elongation factor Tu ERECTA     Receptor Protein Kinase EMS      Ethyl Methanesulfonate; a chemical mutagen ETI       Effector-triggered immunity flg22      flagellin conserved domain of 22 amino acids FLS2      Flagellin-Sensitive 2 H.a. Noco2     Hyaloperonospora arabidopsidis Noco2 HopAI 1     an avirulence protein from Psudomonas syringae LysM      Lysine-motif protein LRR      Leucine-rich repeat MAPK      Mitogen-Activated Protein Kinase MAP2K      MAP kinase kinase MAP3K      MAP kinase kinase kinase MEKK      a MAP3K MKK      MAP kinase kinase MPK      MAP kinase NACK1      NPK1-activating kinesin-like protein 1 NADPH     Nicotinamide adenine dinucleotide phosphate NAG      N-acetyl-D-glucosamine NB       Nucleotide Binding NDR1      Non-race-specific Disease Resistance 1 NPK1      Nucleus- and Phragmoplast- localized kinase 1 NQK1      a MAP2K in tobacco; orthologue of AtMKK6 NRK1      a MAPK in tobacco; downstream of NPK1 and                                      NQK1 OGs      Oligogalacturonides Os       Oryza sativa  xi  PAD      Phytoalexin Deficient PAMP      Pathogen-Associated Molecular Pattern PCR      Polymerase Chain Reaction PR gene     Pathogenesis-Related gene PRR      Pattern Recognition Receptor PBL27      PBS1-Like 27 PBS1      AvrPphB Susceptible 1 PTI       PAMP-triggered immunity Pto       Pseudomonas syringae pv tomato R protein     Resistance protein RBOH      Respiratory Burst Oxidase Homolog RIN4      RPM1-Interacting Protein 4 RLCK      Receptor-Like Cytoplasmic Kinase RLK      Receptor-Like Kinase RNA      Ribonucleic acid ROS      Reactive Oxygen Species RPM1           Resistance to Pseudomonas syringae pv                          Maculicola RPS2      Resistant to Pseudomonas syringae 2 SA       Salicylic acid SUMM      Suppressor of mkk1 mkk2 TIR       Toll Interleukin-1 Receptor TTSS      Type Three Secretion System Yoda      an Arabidopsis MAPK    xii  Acknowledgements  This project was done with a lot of assistance from my colleagues and friends. First, I would like to thank my supervisor, Dr. Yuelin Zhang. He gave me this opportunity to work in his lab and he is so patient to guide and support me in my projects. The advices he provided are extremely valuable to me and facilitate to build up my scientific career. I would like to thank my committee members, Dr. Xin Li and Dr. Jae-Hyeok Lee, for their encouragement and suggestions during my committee meetings.  I would like to thank previous lab member Dr. Pingtao Ding for his training and guiding at the start of my projects.  I would also like to appreciate all the members in Zhang lab and Li lab, who give me a lot of help not only in my work but also in my daily life. Yanan Liu, Tongjun Sun and Yuli Ding always gave me helpful suggestions for my project. Yukino Nitta helped me to revise my thesis. I collaborated with Fang Gao to finish the work in manuscript (Chapter 2), which will be used in her thesis. I want to thank Botany Department for offering me TA positions. I gained a lot of teaching skills from this job. Finally, I want to thank my family. They support my life in Vancouver and they always encourage me to pursue things that I want to do. I really appreciate for their patience and understanding.  1  Chapter 1 Introduction 1.1 Plant immunity Living in an environment with diverse microbes, plants are challenged by pathogens all the time. Every year, there are a great number of crops affected by pathogen infections. This causes large impacts on human activity and economic development. Therefore, protection of crops against pathogens becomes an important topic widely studied by researchers.  Most pathogens have evolved to access the plant interior, either by penetrating the leaf or root surface directly or by entering through open areas on the plants, such as natural openings like stomata and wounds. Fungi can extend hyphae through plant cells, while nematodes and aphids can insert a stylet into plant cells directly. During infection, pathogens can secret effectors into plants to enhance their fitness (Jones and Dangl 2006).  However, plants have also evolved strategies to protect themselves. Unlike animals, plants do not have the adaptive immune system. Instead, they rely on their innate immunity to combat pathogen infections. There are two types of innate immune responses in plants: pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered Immunity (ETI). 1.2 PAMP-triggered immunity PAMP- triggered immunity acts as the first layer in the plant immune system. The pattern recognition receptors (PRRs), which localize on the plasma membrane, can perceive conserved microbial components collectively known as PAMPs. Some examples of well characterized PAMPs are flagellin, EF-Tu and chitin, while the corresponding pattern recognition receptors are FLS2, EFR and CERK1.   2  1.2.1 PRRs and PAMPs 1.2.1.1 FLS2 and flg22 FLAGELLIN SENSING 2 (FLS2) has been well studied as a receptor-like   kinase (RLK), which contains an extracellular domain with 28 Leucine-Rich Repeats (LRRs), a transmembrane domain and an intracellular Ser/Thr protein kinase domain (Gomez-Gomez and Boller 2000). FLS2 can perceive the conserved 22-amino acid epitope flg22 on bacterial flagellin (Felix, Duran et al. 1999).  Upon flg22 perception, FLS2 can form a complex with another RLK, BRASSINOSTEROID INSENSITIVE1 (BRI1)-ASSOCIATED RECEPTOR KINASE1 (BAK1), which is also an important component involved in plant immunity (Chinchilla, Zipfel et al. 2007). A structural study revealed that the N terminus of flg22 binds to the surface of the FLS2 extracellular domain spanning 14 LRRs (LRR3-16 from N terminus of FLS2). The flg22-bound FLS2 ectodomain directly interacts with the BAK1 ectodomain with the C-terminus of flg22 stabilizing the dimerization of FLS2-BAK1, which acts as a molecular glue between two ectodomains (Sun, Li et al. 2013).  1.2.1.2 EFR and EF-Tu The fls2 mutant shows insensitivity to flg22 treatment, while when treated with crude bacterial extracts, defense responses were still detected. This indicates that there are additional PAMPs in bacteria, other than flg22. To identify these PAMPs, the crude extracts of E.coli GI826 (FliC-), which lacks flagellin, was used to isolate and purify new elicitors. By analyzing the PAMP activity of each fraction of crude bacterial extracts, elongation factor-Tu (EF-Tu) was identified as a PAMP. The EF-Tu sequences are highly conserved amongst various bacterial species. The elicitor activity was shown in the 18 amino acids at the N terminus of EF-Tu. Thus, this synthetic peptide named elf18 was widely used as a PAMP (Kunze, Zipfel et al. 2004).  3  To identify the receptor of EF-Tu, a screen of T-DNA insertion lines of RLKs which were induced by either flg22 or EF-Tu was performed. EF-Tu Receptor (EFR) was identified as its T-DNA insertion line was insensitive to EF-Tu treatment. To further confirm that EFR is specific to EF-Tu, a complementation assay was done in the tobacco system. Nicotiana benthamiana lacks the ability to perceive EF-Tu, but showed defense response when EFR was transient transformed (Zipfel, Kunze et al. 2006).  1.2.1.3 CERK1 and chitin  Chitin, a polymer of N-acetyl-D-glucosamine (NAG), is a major component of fungal cell wall and acts as a PAMP. CERK1 (Miya, Albert et al. 2007) and LysM RLK1 (Wan, Zhang et al. 2008) were identified as the receptor of chitin in Arabidopsis. CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) is a plasma membrane localized protein with three lysine motif (LysM) domains, which were found in a variety of enzymes involved in bacterial cell wall degradation and have a general ability to bind peptidoglycan (Visweswaran, Leenhouts et al. 2014). CERK1 contains an intracellular Ser/Thr kinase domain with autophosphorylation / myelin basic protein (MBP) kinase activity (Miya, Albert et al. 2007). A structural study revealed the interaction between chitin and CERK1. The dimerization of CERK1 ectodomain was induced by chitin, which acts as a bivalent ligand and this dimerization is critical for CERK1 activation (Liu, Liu et al. 2012). 1.2.2 Downstream defense responses Upon perception of PAMPs by PRRs, several chemical and physical defense mechanisms are activated in plants to protect against pathogen infection, such as callose deposition, ROS production, Ca2+ influx and MAPK activation. 1.2.2.1 Callose deposition Callose is a high-molecular weight (1,3)-β-glucan cell wall polymer. Callose  4  deposition, as a physical barrier between the plasma membrane and cell wall, can be induced in response to pathogen attack to prevent invading pathogens at the infection site (Nishimura, Stein et al. 2003). Therefore, callose deposition has been an effective response marker for studying signaling pathways involved in plant immunity.  1.2.2.2 ROS production Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), are produced as signaling messengers in stress response. ROS can be produced in the chloroplast, peroxisomes and mitochondria. In plants, ROS are proposed to act as antimicrobial and cross-linkers of the plant cell wall to prevent pathogen invasion (Lamb and Dixon 1997). ROS are produced by NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (NADPH) oxidases, which belong to the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) family. ROS production is considered one of the early responses induced by PAMPs in plant immunity. Accumulation of ROS can be observed after flg22 and elf18 treatment, while in fls2 or efr mutants, no ROS accumulation can be detected after treatment with flg22 or elf18 (Zipfel, Kunze et al. 2006).  1.2.2.3 MAPK cascade activation Mitogen-activated protein kinase (MAPK) cascades amplify signals from upstream stimuli through three types of phosphorylated kinases. The sequential phosphorylation starts from the MAP kinase kinase kinases (MAP3Ks), which can phosphorylate two Ser/Thr residues in the activation loop (Ser/Thr-X3-5-Ser/Thr motif) of MAP kinase kinases (MAP2Ks). Then the MAP2Ks phosphorylate a Thr and Tyr residue in the Thr-X-Tyr activation loop of MAP kinases (MAPKs). The activated MAPKs then target downstream proteins to regulate biological processes (Meng and Zhang 2013). In Arabidopsis, there are approximately 60 MAP3Ks, 10 MAP2Ks and 20 MAPKs based on the genome sequence (Asai,  5  Tena et al. 2002). An immunity-related MAPK cascade, which consists of MEKK1, MKK1 MKK2 and MPK4, was reported to negatively regulate defense responses (Gao, Liu et al. 2008). The roles of MEKK1, MKK1 MKK2 and MPK4 in plant immunity were revealed by their corresponding mutants. Both mekk1 and mpk4 single mutants show dwarfism and high H2O2 accumulation as well as extensive cell death and constitutive PR genes expression (Petersen, Brodersen et al. 2000, Ichimura, Casais et al. 2006, Gao, Liu et al. 2008). MKK1 and MKK2 are functionally redundant and the mkk1mkk2 double mutants showed similar immune phenotypes as mekk1 and mpk4, exhibiting high levels of H2O2 accumulation, extensive cell death and constitutive expression of PR genes (Gao, Liu et al. 2008, Qiu, Fiil et al. 2008). Additionally, the interactions between MEKK1, MKK1/MKK2 and MPK4 were examined using bimolecular fluorescence complementation (BiFC) assay. Interaction between MEKK1 and MKK1/MKK2 were detected on the plasma membrane, while MPK4 and MKK1/MKK2 was found to interact in the nucleus and plasma membrane(Gao, Liu et al. 2008).  To investigate the mechanism how MEKK1-MKK1 MKK2-MPK4 cascade regulates plant immunity, a suppressor screen of mkk1 mkk2 was carried out. Suppressors of mkk1 mkk2 (SUMMs) were identified through restored sizes of mkk1mkk2. SUMM2 encodes a coiled-coil NB-LRR type R protein and the mutation in SUMM2 can suppress cell death, accumulation of H2O2 and elevated PR genes expression in mkk1 mkk2. The constitutively active defense responses in mekk1 and mpk4 are also largely reduced by summ2. The pathogenic effector HopAI1 from Pseudomonas syringae was found to target MPK4 and block its kinase activity. Then it activates SUMM2-mediated defense responses.(Zhang, Wu et al. 2012) Another mkk1/mkk2 suppressor, SUMM1, was also identified and it encodes  6  MEKK2 which is a MAP3K. The constitutive activation of defense responses in mekk1 mkk1/mkk2 and mpk4 is largely blocked by the mutation in MEKK2. MPK4 and MEKK2 interact in the Yeast-two hybrid and co-immunoprecipitation (Co-IP) assays. Overexpressing MEKK2 also causes activation of cell death and defense responses. However, defense responses are not activated when MEKK2 was overexpressed in the summ2 mutant background, indicating that constitutive defense responses in the MEKK2 overexpression lines are dependent on SUMM2 (Kong, Qu et al. 2012). Therefore, the MEKK1-MKK1/MKK2-MPK4 cascade negatively regulated defense responses by inhibiting MEKK2, which triggers SUMM2-mediated immune responses (Kong, Qu et al. 2012, Zhang, Wu et al. 2012). Another MAPK cascade consisting of an unknown MAP3K, MKK4/MKK5 and MPK3/MPK6 was reported to act upstream of PHYTOALXIN DEFICIENT 2 (PAD2) and PAD3 to positively regulate biosynthesis of the phytoalexin camalexin (Ren, Liu et al. 2008). Reduced resistance against Botrytis cinerea and compromised camalexin accumulation were shown in mpk3 and mpk6 mutants, supporting the positive role of this cascade in camalexin biosynthesis in plants when challenged by pathogens (Ren, Liu et al. 2008). 1.2.3 Signaling components downstream of PAMP receptors  Receptor-like kinases (RLKs) are transmembrane proteins with N-terminal extracellular domains and C-terminal intracellular kinase domains. Plants have evolved a large repertoire of RLKs that form the largest family of plant membrane receptors with 610 members in Arabidopsis and 1,100 members in rice (Shiu, Karlowski et al. 2004). Some of the well-studied PAMP receptors are RLKs. Plants have also evolved a large number of receptor-like cytoplasmic kinases (RLCKs), with 147 members in Arabidopsis and 379 members in rice (Shiu and Bleecker 2001, Vij, Giri et al. 2008). RLCKs are potentially anchored to the  7  plasma membrane through N-terminal putative myristoylation or palmitoylation motif. Lacking extracellular domain and transmembrane domain, RLCKs exclusively contain a Ser/Thr-special cytoplasmic kinase domain and more likely function to transduce signal rather than ligand perception (Lin, Ma et al. 2013).  One well studied RLCK is BOTRYTIS-INDUCED KINASE 1 (BIK1). BIK1 was identified as an early induced gene in response to infection by Botrytis cinerea. BIK1 was shown to play important roles in mediating PTI activated by multiple PAMPs. Based on in vitro assays, a general model of BIK1 in flagellin signaling was proposed (Lu, Wu et al. 2010, Zhang, Li et al. 2010). Upon flagellin binding to FLS2, flg22 induces FLS2 and BAK1 association and phosphorylation. The activated BAK1 phosphorylates BIK1, which in turn transphosphorylates the FLS2-BAK1 complex. The active BIK1 is likely disassociated from the FLS2-BAK1 complex to activate downstream intracellular signaling (Lu, Wu et al. 2010, Zhang, Li et al. 2010). Recent studies showed that activated BIK1 directly phosphorylated RBOHD, which leads to ROS production (Kadota, Sklenar et al. 2014, Li, Li et al. 2014). These evidences support that BIK1 is a critical regulator in the pathway from PAMP perception to downstream signal transduction.  PBS1-LIKE 27 (PBL27) was identified as an Arabidopsis orthologue of OsRLCK185 which was reported to interact with OsCERK1. Knockout mutant of PBL27 resulted in the reduction of chitin-induced callose deposition and resistance against fungal infection. CERK1 was found to preferentially phosphorylate PBL27 rather than BIK1, while phosphorylation of PBL27 was not induced by flg22 treatment, suggesting that the signaling mediated by PBL27 is specific to CERK1 (Shinya, Yamaguchi et al. 2014). 1.3 Effector-triggered susceptibility Successful pathogens deliver effectors to inhibit PTI responses using the Type Three Secretion System (TTSS) to promote bacterial growth. Some  8  effectors have been shown to target cell components that are involved in immune responses. Two unrelated effectors secreted from Pseudomonas syringae AvrB and AvrRpm1 suppress PTI by promoting phosphorylation of RIN4, which acts as a negative regulator in PTI. Additionally, a third effector AvrRpt2, which is a cysteine protease, was reported to cleave RIN4 (Mackey, Holt et al. 2002, Axtell and Staskawicz 2003, Mackey, Belkhadir et al. 2003, Kim, Desveaux et al. 2005, Chung, da Cunha et al. 2011, Liu, Elmore et al. 2011). Pseudomonas syringae can secret other two effectors, AvrPto and AvrPtoB, to enhance their virulence by targeting BAK1 and disrupting flagellin-induced FLS2-BAK1 complex (Shan, He et al. 2008).   1.4 Effector-triggered immunity Pathogen effectors can be perceived by resistance (R) proteins in plant. Most R genes encode Nucleotide Binding (NB)-Leucine Rich Repeat (LRR) proteins. After perception of effectors, R proteins usually trigger strong defense responses, which is effector-triggered immunity (ETI) (Jones and Dangl 2006).  Plant NB-LRR R proteins can be divided into two groups based on the sequence of N-terminal regions: Coiled-Coil (CC) NB-LRR and Toll Interleukin-1 Receptor (TIR) NB-LRR. The N-terminal domains may allow R proteins to interact with different targets in order to activate various signaling pathways. Effectors are recognized by R proteins either through directly binding to receptors or facilitation of an accessory protein (Jones and Dangl 2006). For example, phosphorylation of RIN4 by AvrB or AvrRpm1 can be perceived by the coiled-coil R protein RPM1 to activate RPM1-mediated immune responses (Chung, da Cunha et al. 2011, Liu, Elmore et al. 2011). The cleavage of RIN4 by AvrRpt2 can be sensed by another coiled-coil R protein RPS2, which activates RPS2-mediated defense responses (Axtell and Staskawicz 2003, Mackey, Belkhadir et al. 2003).   9  1.4.1 R protein signaling Several genes required for R protein-mediated signaling pathway have been identified. Loss of function of NON-RACE-SPECIFIC DISEASE RESISTANCE 1 (NDR1) shows susceptibility to Pseudomonas syringae pv. tomato strain DC3000 carrying avirulence genes, avrB, avrRpm1, avrRpt2 and avrPph3, which are recognized by coiled-coil NB-LRR proteins (Century, Holub et al. 1995), suggesting that NDR1 is required for the signaling pathway activated by CC-NB-LRR R protein. ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN DEFICIENT 4 (PAD4) were identified as positive regulators of the TIR-NB-LRR protein-mediated signaling pathway (Parker, Holub et al. 1996, Falk, Feys et al. 1999, Jirage, Tootle et al. 1999). Both EDS1 and PAD4 encode lipase-like proteins and they act as positive regulators in SA production (Jirage, Tootle et al. 1999, Feys, Moisan et al. 2001). EDS1 and PAD4 interact with each other and the interaction is required for TIR-NB-LRR R protein-mediated disease resistance (Feys, Moisan et al. 2001, Wagner, Stuttmann et al. 2013).  1.5 Roles of ANPs in plant development and immunity  1.5.1 ANPs regulate cytokinesis process  ARABIDOPSIS NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE 1 RELATED PROTEIN KINSES (ANPs) are a subfamily of MAP3Ks which includes three members, ANP1, ANP2 and ANP3. They were originally identified as orthologues of NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE 1 (NPK1) in tobacco (Nakashima, Hirano et al. 1998).  NPK1 localizes to the phragmoplast during cytokinesis and cytokinesis defective phenotypes such as multinucleate cells with incomplete cell plate and inhibition of phragmoplast expansion were observed when a kinase-negative version of NPK1 was expressed (Nishihama, Ishikawa et al. 2001). NPK1 regulates cytokinesis in tobacco through a MAPK cascade, which includes NPK1  10  (MAP3K), NQK1 (MAP2K) and NRK1 (MAPK). This cascade is activated by binding of kinesin-like proteins NPK1-activating kinesin-like protein 1 (NACK1) and NACK2 to NPK1 at the late mitotic phase of the cell cycle (Nishihama, Soyano et al. 2002, Takahashi, Soyano et al. 2004). Arabidopsis anp1, anp2 and anp3 single knockout mutants have no obvious development phenotypes. The anp2 anp3 double mutant has reduced plant size (Krysan, Jester et al. 2002). The hypocotyls and petals in anp2 anp3 consist of a number of irregularly shaped cells with reduced elongation and radial swelling. No developmental defects were found in anp1 anp2, while only abnormal floral organs were showed in anp1 anp3. The anp1 anp2 anp3 triple mutant cannot be obtained by crossing, indicating that these three genes are collectively essential in Arabidopsis (Krysan, Jester et al. 2002).  1.5.2 Roles of ANPs in immune responses Several stress related genes, including pathogen response related genes and oxidative stress related genes, were found to be upregulated in the anp2 anp3 double mutant based on microarray data (Krysan, Jester et al. 2002). To clarify the roles of ANPs in plant defense, the conditional silencing plants were generated by expressing ANP3- and ANP1- specific artificial microRNAs under the control of a β-estradiol inducible promoter in the anp1 anp2 or anp2 anp3 double mutant background (Savatin, Bisceglia et al. 2014). The ROS production was significantly reduced in the anp2 anp3 double mutant and conditional triple mutants with the treatment of DANGER- ASSOCIATED MOLECULAR PATTERN (DAMP) oligogalacturonides (OGs) and PAMP elicitor elf18. In contrast, transgenic lines overexpressing ANP1 or ANP3 under the control of CaMV 35S promoter showed increased induction of ROS in response to OGs and efl18. In addition, a small reduction of OGs-induced MPK3/6 phosphorylation was also observed when the functions of ANPs were compromised. These data suggest  11  that ANPs play a positive role in elicitor-induced defense responses (Savatin, Bisceglia et al. 2014). 1.5.3 MKK6 and MPK4 function downstream of ANPs to regulate cytokinesis A mutation in Arabidopsis MKK6, which is the orthologue of NQK1 in tobacoo, causes dwarfism and large multinucleate cells with incomplete cell wall (Soyano, Nishihama et al. 2003), which is reminiscent of the mutant phenotype of anps. In addition, MKK6 was shown to localize at the equatorial plane of the phragmoplast (Takahashi, Soyano et al. 2010). MKK6 interacts with ANP3 in the nucleus in the BiFC assay (Zeng, Chen et al. 2011). In the presence of AtNACK1, ANPs can activate MKK6 in yeast (Takahashi, Soyano et al. 2010). These data suggest that MKK6 acts as the downstream MAP2K of ANPs in the regulation of cytokinesis.  Yeast two-hybrid analysis using the full set of AtMAP2Ks and AtMAPKs revealed that MKK6 can interact with MPK4, MPK11, MPK6 and MPK13 (Lee, Huh et al. 2008). These interactions were further confirmed by BiFC in Arabidopsis protoplasts (Zeng, Chen et al. 2011). In-gel kinase assays revealed MPK4 can be phosphorylated by MKK6 (Takahashi, Soyano et al. 2010). In a separate study, only MPK4 was strongly phosphorylated by constitutively active (CA) version of MKK6 (Zeng, Chen et al. 2011). These data suggest that MPK4 functions downstream of MKK6. Similar to mkk6 mutant, mpk4 mutant plant shows a dwarf phenotype and exhibits abnormal cell grown patterns such as branching of root hairs and swelling of diffusely growing epidermal cells. These phenotypes are observed in the anp2 anp3 double mutant (Beck, Komis et al. 2011). The cortical microtubule organization in the hypocotyl epidermal cells of anp2 anp3 and mpk4 was disoriented to form random net-like arrays instead of parallel arrays at the cell cortex (Beck, Komis et al. 2010). The microspore mother cells of mkp4 failed to form a normal intersporal callose wall after male meiosis and the multinucleate  12  microspores undergo mitotic cytokinesis leading to enlarged pollen grains, which is reminiscent of the mkk6 phenotype as well (Zeng, Chen et al. 2011). The similarities in anp2 anp3, mkk6 and mpk4 mutant phenotypes also suggest that MPK4 may form a MAPK cascade together with ANPs and MKK6 to regulate cytokinesis in Arabidopsis.  Taking all of these evidences together, we generate a hypothesis that ANP2/3-MKK6-MPK4 can form a cascade to regulate defense responses.     13  Chapter 2 Arabidopsis MKK6 functions in two parallel MAP kinase cascades that negatively regulate plant immunity 2.1 Summary Arabidopsis MPK4 is a component of two independent MAP kinase cascades and functions in regulating development as well as plant defense. The MEKK1-MKK1/MKK2-MPK4 cascade inhibits activation of resistance protein-mediated defense responses mediated by SUMM2 and is also required for basal resistance, whereas the ANPs-MKK6-MPK4 cascade plays essential roles in cytokinesis. Here we report that MKK6 also functions together with MEKK1 and MPK4 to prevent activation of SUMM2-mediated immunity and the ANP2/ANP3-MKK6-MPK4 cascade negatively regulate a defense pathway that is independent of SUMM2. A gain-of-function mutant identified from a suppressor screen of mkk1mkk2 suppresses mkk1mkk2 but not mekk1 or mpk4 autoimmune phenotypes, suggesting that MKK6 shares similar functions as MKK1/MKK2 and works together with MEKK1 and MPK4 to inhibit activation of SUMM2-mediated defense responses. Interestingly, loss of MKK6 or ANP2 and ANP3 also causes constitutive activation of plant defense responses. The autoimmune phenotype in mkk6 and anp2 anp3 mutant plants can be largely suppressed by a constitutively active mutant of MPK4. Furthermore, constitutive defense responses in anp2 anp3 is dependent on the defense regulator PAD4, but not SUMM2, suggesting that the ANP2/ANP3-MKK6-MPK4 cascade negatively regulates a SUMM2-independent defense pathway.     14  2.2 Introduction In Arabidopsis, there are 20 Mitogen-activated protein kinases (MAPKs), 10 MAPK kinases (MAPKKs) and about 60 MAPK kinase kinases (MAPKKKs) (MAPK-Group 2002). They work in combinations to form distinct MAP kinase cascades that play diverse roles in plant development and stress signaling (Rodriguez, Petersen et al. 2010, Meng and Zhang 2013). Several MAP kinase cascades including Yoda- MKK4/MKK5- MPK3/MPK6, MEKK1- MKK1/MKK2- MPK4 and ANPs (Arabidopsis NPK1-related Protein Kinases)- MKK6- MPK4 have been studied extensively. Arabidopsis MKK4/MKK5 and MPK3/MPK6 function in regulating both development and defense against pathogens. They form a MAP kinase cascade with Yoda to mediate signal transduction from upstream RLKs such as ERECTA and BAK1 to the downstream transcription factors in stomata development (Bergmann and Sack 2007, Meng, Chen et al. 2015). In response to elicitor treatment, the MAP kinase cascade consisting of MKK4/MKK5, MPK3/MPK6 and an unknown MAPKKK are activated (Asai, Tena et al. 2002). This kinase cascade has been shown to play critical roles in the regulation of biosynthesis of ethylene and phytoalexin (Liu and Zhang 2004, Ren, Liu et al. 2008). The MEKK1-MKK1/MKK2-MPK4 cascade is also activated following elicitor treatment (Gao, Liu et al. 2008, Qiu, Zhou et al. 2008). Components of this kinase cascade were originally identified as negative regulators of plant immunity based on the autoimmune phenotypes in the mekk1, mkk1 mkk2 and mpk4 mutants (Petersen, Brodersen et al. 2000, Ichimura, Casais et al. 2006, Nakagami, Soukupova et al. 2006, Suarez-Rodriguez, Adams-Phillips et al. 2007, Gao, Liu et al. 2008, Qiu, Zhou et al. 2008). Further studies on the suppressor mutants of mkk1 mkk2 showed that autoimmunity in these mutants is caused by activation of the coiled-coil (CC)-nucleotide binding (NB)–Leu-rich repeat (LRR) protein  15  SUMM2 (Zhang, Wu et al. 2012). The autoimmune phenotypes in the mekk1, mkk1 mkk2 and mpk4 mutants are also dependent on MEKK2 (Kong, Qu et al. 2012, Su, Bush et al. 2013), but the mechanism is unclear. In the absence of SUMM2, mekk1 and mkk1 mkk2 mutant plants showed enhanced susceptibility to pathogens, suggesting that the MEKK1-MKK1/MKK2-MPK4 cascade functions in promoting basal resistance against pathogens (Zhang, Wu et al. 2012). Consistently, MPK4 is required for the induction of about 50% genes by flg22 (Frei dit Frey, Garcia et al. 2014). Interestingly, MPK4 also plays a role in the negative regulation of flg22-induced gene expression through phosphorylation of ASR3 (Li, Jiang et al. 2015). From a functional yeast screen, mutations that render Arabidopsis thaliana MAPKs constitutively active have been identified (Berriri, Garcia et al. 2012). The specificity toward known activators and substrates appears to be unchanged in the constitutively active mutants of MAPKs (CA-MPKs). CA-MPK4 transgenic plants accumulate less salicylic acid following pathogen infection and exhibit enhanced susceptibility to pathogens (Berriri, Garcia et al. 2012). Interestingly, effector-triggered immunity specified by the Toll Interleukin-1 Receptor (TIR)–NB–LRR resistance proteins RPS4 and RPP4 were also found to be compromised in CA-MPK4 transgenic plants, suggesting that constitutive activation of MPK4 inhibits resistance mediated by RPS4 and RPP4.  ANP1, ANP2 and ANP3 are three MAPKKKs closely related to NPK1, which is involved in the regulation of cytokinesis in tobacco (Nishihama, Ishikawa et al. 2001). Single mutants of anp1, anp2 and anp3 have no clear phenotypes, whereas the anp2 anp3 double mutant displays abnormal cytokinesis (Krysan, Jester et al. 2002). The anp1 anp2 anp3 triple mutant cannot be obtained because of lethality. In Arabidopsis, MKK6 and MPK4 function downstream of ANPs to regulate cytokinesis (Beck, Komis et al. 2010, Kosetsu, Matsunaga et al.  16  2010, Takahashi, Soyano et al. 2010, Zeng, Chen et al. 2011). Loss of MKK6 or MPK4 leads to severe defects in cytokinesis. In this study, we report that MKK6 also functions together with MEKK1 and MPK4 to negatively regulate SUMM2-mediated immunity and the ANP2/ANP3-MKK6-MPK4 cascade plays a critical role in negative regulation of defense responses independent of SUMM2.  2.3 Results 2.3.1 Characterization of summ4-1D mkk1 mkk2  From a previously described suppressor screen of mkk1 mkk2 (mkk1/2) (Zhang, Wu et al. 2012), we identified summ4-1D that suppresses the dwarf phenotype of mkk1/2 almost completely (Figure 1A).  It was named summ4-1D because the mutation was later found to be dominant. To determine whether constitutive defense responses in mkk1/2 is suppressed by summ4-1D, we checked the expression levels of defense marker genes Pathogenesis-Related 1 (PR1) and PR2 in summ4-1D mkk1/2. As shown in Figure 1B and 1C, constitutive expression of PR1 and PR2 in mkk1/2 is completely suppressed in the summ4-1D mkk1/2 triple mutant. We further tested whether summ4-1D affects pathogen resistance in mkk1/2 by challenging seedlings of summ4-1D mkk1/2 with the oomycete pathogen Hyaloperonospora arabidopsidis (H.a.) Noco2. As shown in Figure 1D, growth of H.a. Noco2 on summ4-1D mkk1/2 was much higher than on mkk1/2 (Figure 1D). These data suggest that the constitutive defense responses in mkk1/2 are suppressed by the summ4-1D mutation.   17    Figure 1 Characterization of summ4-1D mkk1 mkk2. (A) Morphological phenotypes of three-week-old wild type (WT), mkk1 mkk2 and summ4-1D mkk1 mkk2 plants.  (B-C) Expression levels of PR1 (B) and PR2 (C) in WT, mkk1 mkk2 and summ4-1D mkk1 mkk2 seedlings. Values were normalized relative to the expression of ACTIN1. Error bars represent standard deviations from three measurements. (D) Growth of H. a. Noco2 on WT, mkk1 mkk2 and summ4-1D mkk1 mkk2 plants. Error bars represent standard deviations from three replicates. Statistical differences among the samples are labelled with different letters (P < 0.01, one-way ANOVA, n=3).   18  2.3.2 Positional cloning of SUMM4 In the F2 progeny of a cross between wild type and summ4-1D mkk1/2, plants homozygous for mkk1/2 and heterozygous for summ4-1D were identified. These plants had wild type morphology, suggesting that the summ4-1D mutation is dominant. To map the summ4-1D mutation, summ4-1D mkk1/2 was crossed with Landsberg erecta (Ler). Plants that are mkk1/2 homozygous in the F2 population were selected for linkage analysis. Crude mapping showed that the summ4-1D mutation is located between markers K19E20 and MMN10 on chromosome 5 (Figure 2A). To identify the summ4-1D mutation, a genomic DNA library of summ4-1D mkk1/2 was prepared and sequenced. Using single nucleotide polymorphisms identified from the sequence data and progeny of F2 plants homozygous for mkk1/2 and heterozygous for summ4-1D, we further narrowed the summ4-1D mutation to a region between markers 22.8 and 23.5 on chromosome 5 (Figure 2A). Only one mutation, a C-to-T substitution located in the promoter region of MKK6 (At5g56580), was identified in this region (Figure 2A).  To test whether this mutation causes suppression of the mkk1/2 mutant phenotype, a genomic clone of MKK6 carrying the candidate summ4-1D mutation was transformed into plants homozygous for mkk1 and heterozygous for mkk2, as mkk1/2 double mutant is seedling lethal. Transgenic plants homozygous for mkk1 and mkk2 were identified by PCR and they were found to display wild type-like morphology (Figure 2B), suggesting that the mutation in the promoter region of MKK6 is indeed responsible for the suppression of the mkk1 mkk2 mutant phenotypes in summ4-1D mkk1/2.   19    20  Figure 2 Positional cloning of SUMM4. (A) Map position and the mutation in summ4-1D.  (B) Morphology of three-week-old mkk1 mkk2 plants expressing MKK6 driven by its native promoter containing the summ4-1D mutation. Three independent transgenic lines are shown.  (C) MKK6 expression levels in WT, mkk1 mkk2, summ2-8 mkk1 mkk2, summ4-1D mkk1mkk2 and summ4-1D seedlings. Values were normalized relative to the expression of ACTIN1. Error bars represent standard deviations from three measurements.   21  2.3.3 The summ4-1D mutation results in elevated expression of MKK6  Since the summ4-1D mutation is in the promoter region of MKK6, we tested whether the expression level of MKK6 is affected. As shown in Figure 2C, the summ4-1D mkk1/2 triple mutant has much higher expression of MKK6 than wild type and mkk1/2. In contrast summ2-8 does not affect the expression of MKK6 in mkk1 mkk2.  Similarly, the expression level of MKK6 is also dramatically increased in the summ4-1D single mutant compared to wild type and mkk1/2, suggesting that the summ4-1D mutation causes increased MKK6 expression. 2.3.4 summ4-1D does not suppress the autoimmune phenotypes of mekk1 and mpk4  Since MEKK1 functions upstream of MKK1/MKK2, we generated the summ4-1D mekk1-1 double mutant to test whether the mekk1 mutant phenotype can be suppressed by summ4-1D. As shown in Figure 3A, summ4-1D mekk1-1 has similar dwarf morphology as mekk1-1. The expression levels of PR1 and PR2 in the double mutant are also comparable to those in mekk1-1 (Figure 3B and 3C), suggesting that summ4-1D cannot suppress the constitutive defense responses in mekk1-1. We also generated the summ4-1D mpk4-3 double mutant to test whether the mpk4 mutant phenotype can be suppressed by summ4-1D. Morphologically the summ4-1D mpk4-3 double mutant is indistinguishable from mpk4-3 (Figure 3D). Analysis of the expression levels of PR1 and PR2 in summ4-1D mpk4-3 showed that they are also similar to those in mpk4-3 (Figure 3E and 3F), indicating that the autoimmunity phenotype in mpk4-3 cannot be suppressed by the summ4-1D mutation.   22    23  Figure 3 summ4-1D does not suppress the autoimmune phenotypes of mekk1 and mpk4. (A) Morphology of three-week-old WT, summ4-1D, mekk1-1 and summ4-1D mekk1-1 plants. (B-C) Expression levels of PR1 (B) and PR2 (C) in WT, mekk1-1 and summ4-1D mekk1-1 seedlings. Values were normalized relative to the expression of ACTIN1. Error bars represent standard deviations from three measurements. (D) Morphology of three-week-old WT, summ4-1D, mpk4-3 and summ4-1D mpk4-3 plants. (E-F) Expression levels of PR1 (E) and PR2 (F) in WT, mekk1-1 and summ4-1D mekk1-1 seedlings. Values were normalized relative to the expression of ACTIN1. Error bars represent standard deviations from three measurements.     24  2.3.5 MKK6 interacts with MEKK1 and MPK4  To test whether MKK6 interacts with MEKK1 and MPK4, split luciferase complementation assays were conducted using constructs expressing MKK6 fused to the C-terminal domain of luciferase (MKK6CLuc) and MEKK1 and MPK4 fused to the N-terminal domain of luciferase (MEKK1NLuc and MPK4NLuc) under a 35S promoter.  If MKK6 associates with MEKK1 or MPK4, a functional luciferase complex would be formed. Consistent with a previous report that MPK4 interacts with MKK6 in bifluorescence complementation assays, strong luciferase activity was observed when MKK6CLuc and MPK4NLuc were co-expressed in Nicotiana (N.) benthamiana (Figure 4). Luciferase activity was also observed when MKK6CLuc and MEKK1NLuc were co-expressed in N. benthamiana, despite at lower levels (Figure 4). These data suggest that MKK6 interacts with both MEKK1 and MPK4 in vivo.   25     Figure 4 Interactions between MKK6 and MEKK1 or MPK4. Quantitative analysis of luciferase activity, represent in Relative Light Units (RLU) of split luciferase complementation assay. Error bars represent standard deviations from eighet replicates. Statistical differences among the samples are labelled with different letters (P < 0.05, one-way ANOVA, n=8).   26  2.3.6 PR1 and PR2 are constitutively expressed in mkk6 mutant plants To test whether defense responses are activated in mkk6 mutant plants, we analyzed the expression of PR1 and PR2 in mkk6-2. As shown in Figure 5B and 5C, both PR1 and PR2 are constitutive expressed in mkk6-2. To determine whether constitutive activation of defense gene expression is caused by reduced MPK4 activity, we crossed mkk6-2 with a transgenic line expressing the MPK4D198G/E202A (CA-MPK4) mutant that is constitutively active and obtained the mkk6-2 CA-MPK4 double mutant. Quantitative RT-PCR analysis showed that the constitutive expression of PR1 and PR2 in mkk6-2 is largely blocked by the CA-MPK4 transgene (Figure 5E and 5F), suggesting that MPK4 functions downstream of MKK6.     27   Figure 5 Expression of PR genes in mkk6 and CA-MPK4 mkk6. (A) Morphology of three-week-old WT and mkk6 plants. (B-C) PR1 and PR2 expression levels in WT and mkk6 seedlings.  (D) Morphology of three-week-old WT, mkk6 and mkk6 CA MPK4 plants. (E-F) PR1 and PR2 expression levels in WT, mkk6 and CA-MPK4 mkk6 seedlings. Values were normalized relative to the expression of ACTIN1. Error bars represent standard deviations from three measurements.   28  2.3.7 Defense responses are constitutively activated in the anp2 anp3 double mutant ANPs have previously been shown to interact with MKK6 and function upstream of MKK6 in regulating cytokinesis (Krysan, Jester et al. 2002, Beck, Komis et al. 2010, Kosetsu, Matsunaga et al. 2010, Takahashi, Soyano et al. 2010). Since microarray analysis showed that stress-related genes are up-regulated in the anp2 anp3 double mutant (Krysan, Jester et al. 2002), we examined whether ANP2 and ANP3 are involved in negative regulation of plant immunity. Compared to wild type and the anp2-2 and anp3-3 single mutants, the anp2-2 anp3-3 double mutant exhibits dwarf morphology (Figure 6A). Both PR1 and PR2 are constitutively expressed in the anp2-2 anp3-3 double mutant, but not in the anp2-2 and anp3-3 single mutants (Figure 6B and 6C). To determine whether anp2-2 anp3-3 exhibits enhanced pathogen resistance, it was challenged with H. a. Noco2. As shown in Figure 6D, H. a. Noco2 growth is greatly reduced in the anp2-2 anp3-3 double mutant compared to those in the wild type and the single mutants, suggesting that ANP2 and ANP3 function redundantly in negative regulation of plant defense responses.   29    Figure 6 Characterization of the anp2 anp3 double mutant. (A) Morphology of three-week-old WT, anp2-2, anp3-3 and anp2-2 anp3-3 plants. (B-C) PR1 (B) and PR2 (C) expression levels in WT, anp2-2, anp3-3 and anp2-2 anp3-3 seedlings.  (D) H. a. Noco2 growth on WT, anp2-2, anp3-3 and anp2-2 anp3-3 plants. Statistical differences among the samples are labelled with different letters (P <0.01, one-way ANOVA, n=3)   30  2.3.8 The autoimmune phenotype of anp2 anp3 can be partially suppressed by the CA-MPK4 mutant To test whether the autoimmune phenotype in anp2-2 anp3-3 is due to reduced activity of MPK4, the anp2-2 anp3-3 double mutant was crossed with a transgenic line expressing the CA-MPK4 mutant to obtain the anp2 anp3 CA- MPK4 triple mutant. As shown in Figure 7A, the dwarf morphology of anp2 anp3 is partially suppressed by the CA-MPK4 mutant. Analysis of PR gene expression showed that the expression levels of both PR1 and PR2 are also lower in the anp2 anp3 CA-MPK4 triple mutant (Figure 7B and 7C). In addition, growth of H. a. Noco2 is much higher in the triple mutant than in the anp2 anp3 double mutant (Figure 7D). These data suggest that ANP2/ANP3 function upstream of MPK4.    31    Figure 7 CA-MPK4 partially blocks the constitutive defense responses in anp2-2 anp3-3. (A) Morphology of three-week-old WT, anp2-2 anp3-3 and CA-MPK4 anp2-2 anp3-3 plants.  (B-C) PR1 (B) and PR2 (C) expression levels in WT, anp2-2 anp3-3 and CA-MPK4 anp2-2 anp3-3 seedlings.  (D) H. a. Noco2 growth on WT, anp2-2 anp3-3 and CA-MPK4 anp2-2 anp3-3 plants. Statistical differences among the samples are labelled with different letters (P <0.01, one-way ANOVA, n=3).     32  2.3.9 Constitutive defense responses in anp2 anp3 are independent of SUMM2 As constitutive defense responses in mpk4 are largely dependent on SUMM2, we tested whether the SUMM2-dependent defense pathway is activated in anp2 anp3. The anp2-2 anp3-3 summ2-8 triple mutant was obtained by crossing summ2-8 into anp2-2 anp3-3. As shown in Figure 8A, summ2-8 has no effects on the morphology of anp2-2 anp3-3. In addition, summ2-8 has no effects on the expression of PR1 (Figure 8B) and PR2 (Figure 8C) and resistance to H. a. Noco2 (Figure 8D), suggesting that the autoimmune phenotype of anp2 anp3 is independent of SUMM2.  2.3.10 PAD4 is partially required for the autoimmune phenotype of anp2 anp3   Constitutive activation of MPK4 was previously shown to compromise effector-triggered immunity specified by the TIR-NB-LRR resistance proteins RPS4 and RPP4. To test whether resistance mediated by TIR-NB-LRR proteins is activated in anp2 anp3, we crossed a loss-of-function mutant of PAD4, which is required for resistance mediated by TIR-NB-LRR proteins (Glazebrook, Rogers et al. 1996, Feys, Moisan et al. 2001), into anp2-2 anp3-3. As shown in Figure 8E, the pad4-1 mutation partially suppresses the dwarf morphology of anp2-2 anp3-3. Elevated expression levels of PR1 and PR2 in anp2-2 anp3-3 are almost completely suppressed in the anp2-2 anp3-3 pad4-1 triple mutant (Figure 8F and 8G). Furthermore, the enhanced resistance to H. a. Noco2 in anp2 anp3 is also abolished in the anp2 anp3 pad4-3 triple mutant (Figure 8H). These data suggest that the autoimmune phenotype of anp2-2 anp3-3 is partially dependent on PAD4.   33     34   Figure 8 Constitutive defense responses in anp2-2 anp3-3 are independent of SUMM2 but dependent on PAD4. (A) Morphology of three-week-old WT, anp2-2 anp3-3 and summ2-8 anp2-2 anp3-3 plants.  (B-C) PR1 (B) and PR2 (C) expression levels in WT, anp2-2 anp3-3 and summ2-8 anp2-2 anp3-3 seedlings.  (D) H. a. Noco2 growth on WT, anp2-2 anp3-3 and summ2-8 anp2-2 anp3-3 plants. Statistical differences among the samples are labelled with different letters (P <0.01, one-way ANOVA, n=3). (E) Morphology of three-week-old WT, anp2-2 anp3-3 and pad4-1 anp2-2 anp3-3 plants.  (F-G) PR1 (F) and PR2 (G) expression levels in WT, anp2-2 anp3-3 and pad4-1 anp2-2 anp3-3 seedlings.  (H) H. a. Noco2 growth on WT, anp2-2 anp3-3 and pad4-1 anp2-2 anp3-3 plants. Statistical differences are labelled with different letters (P <0.01, one-way ANOVA, n=3)  35  2.3.11 anp2-2 anp3-3 is more susceptible to P. syringae pv tomato DC3000 hrcC-  To test whether PTI is affected by loss of function of ANP2 and ANP3, anp2-2, anp3-3 and anp2-2 anp3-3 mutant plants were infiltrated with Pto DC3000 hrcC-, a bacterial strain deficient in delivery of type III effectors and often used as an indicator of PTI. As shown in Figure 9, growth of Pto DC3000 hrcC- is comparable in anp2-2, anp3-3 and wild type, but much higher in the anp2-2 anp3-3 double mutant, suggesting that ANP2 and ANP3 function redundantly in the positive regulation of PTI.    Figure 9 Growth of Pto DC3000 hrcC- in wild type, anp2-2, anp3-3, and anp2-2 anp3-3. Plants were infiltrated with Pto DC3000 hrcC- (OD600 = 0.002). Bacterial growth was measured at day 0 and day 3 by taking leaf discs within the inoculated area. Statistical differences are labelled with different letters (P <0.01, one-way ANOVA, n=8).   36  2.4 Discussion Despite that MEKK1 and MKK1/MKK2 function in the same MAP kinase pathway, the mutant phenotypes of mekk1 and mkk1 mkk2 are not identical (Rodriguez, Petersen et al. 2010). mekk1 knockout mutant plants are much smaller than the mkk1 mkk2 double knockout mutants, suggesting that one or more MKKs may have overlapping functions with MKK1/MKK2. We showed that MKK6 interacts with MEKK1 and MPK4 in split-luciferase assays and elevated expression of MKK6 in the summ4-1D mutant suppresses the autoimmune phenotypes of mkk1 mkk2, but not those in mekk1 and mpk4. These data suggest that MKK6 functions in parallel with MKK1/MKK2 to transduce signals from MEKK1 to MPK4 (Figure 10). Arabidopsis ANPs and MKK6 have previously been shown to function together with MPK4 to regulate cytokinesis (Krysan, Jester et al. 2002, Beck, Komis et al. 2010, Kosetsu, Matsunaga et al. 2010, Takahashi, Soyano et al. 2010). Our data suggest that ANP2/ANP3 and MKK6 also play important roles in plant immunity. anp2 anp3 and mkk6 mutant plants constitutively express PR genes and exhibit enhanced pathogen resistance. These autoimmune phenotypes can be suppressed by a constitutively active MPK4 mutant protein, suggesting that ANP2/ANP3 and MKK6 function together with MPK4 in a MAP kinases cascade to negatively regulate plant defense (Figure 10). Arabidopsis has 60 predicted MAPKKKs, but only 10 MKKs and 20 MAPKs (MAPK-Group 2002), suggesting that some of the MKKs and MAPKs may have multiple functions and can form distinct MAP kinase cascades with different MAPKKKs to regulate different biological processes. This is supported by the diverse roles of MKK4/MKK5 and MPK3/MPK6 in plant defense as well as in development (Meng and Zhang 2013). Our study revealed that MKK6 also has multiple functions. In addition to its roles in cytokinesis, MKK6 is also involved in  37  two MAPK kinase cascades that negatively regulate plant immunity. Both the MEKK1-MKK1/MKK2-MPK4 and ANPs-MKK6-MPK4 cascades lead to activation of MPK4. Mutations in summ2 suppress the autoimmune phenotypes of mekk1 and mkk1 mkk2, but not anp2 anp3, suggesting that these two MAP kinase cascades function independently in the negative regulation of plant immunity (Figure 10). This is consistent with that the mutant phenotypes of mekk1 and mkk1 mkk2 are completely dependent on SUMM2, whereas the constitutive defense responses in mpk4 can only be partially blocked by mutations in summ2 (Zhang, Wu et al. 2012). It is unclear why two kinase cascades both leading to activation of MPK4 cannot compensate each other. Previously it was shown that MEKK1 interacts with MKK1 and MKK2 on the plasma membrane (Gao, Liu et al. 2008), whereas the ANPs-MKK6-MPK4 cascade functions in regulating cytokinesis in the nucleus (Beck, Komis et al. 2010, Kosetsu, Matsunaga et al. 2010, Takahashi, Soyano et al. 2010, Zeng, Chen et al. 2011). It is possible that the MEKK1-MKK1/MKK2-MPK4 and ANPs-MKK6-MPK4 cascades are active in different subcellular localizations to prevent constitutive activation of immune responses.    The mechanism of how the ANP2/ANP3-MKK6-MPK4 cascade negatively regulates plant immunity is unknown. Previously it was shown that expression of a constitutively active MPK4 leads to compromised pathogen resistance mediated by TIR-NB-LRR proteins (Berriri, Garcia et al. 2012). The autoimmune phenotype of anp2 anp3 is dependent on PAD4, which is a critical positive regulator of TIR-NB-LRR protein mediated resistance (Glazebrook, Rogers et al. 1996, Feys, Moisan et al. 2001). It is likely that activation of MPK4 through the ANP2/ANP3-MKK6-MPK4 cascade is required for its functions in negative regulation of immunity mediated by one or more TIR-NB-LRR proteins. Meanwhile, ANPs have been shown to function as positive regulators of  38  elicitor-triggered defense responses and protection against the necrotrophic fungus Botrytis cinerea (Savatin, Bisceglia et al. 2014). Increased growth of Pto DC3000 hrcC- in the anp2-2 anp3-3 double mutant also supports a positive role of ANP2 and ANP3 in PTI. It is likely that components of the ANP2/ANP3-MKK6-MPK4 cascade are targeted by certain pathogens and plants have evolved resistance proteins to sense disruption of this kinase cascade. Similar to protection of the MEKK1-MKK1/ MKK2-MPK4 cascade by the NB-LRR protein SUMM2 (Zhang, Wu et al. 2012), loss of function of ANP2/ANP3, MKK6 or MPK4 would result in activation of immunity mediated by the resistance proteins.    Figure 10 A working model for the roles of MKK6 in plant immunity. MKK6 functions in parallel with MKK1 and MKK2 to form a MAPK cascade to prevent activation of SUMM2-mediated immunity. MKK6 also functions together with ANP2/ANP3 and MPK4 in a separate MAPK cascade that negatively regulates a PAD4-dependent defense pathway.   39  2.5 Methods 2.5.1 Plant materials  The summ4-1D mkk1/2 triple mutant was isolated from an EMS mutagenized M2 population of mkk1/2 (Zhang, Wu et al. 2012). mkk1-1 mkk2-1 (mkk1 mkk2 or mkk1/2), mpk4-3, mekk1-1, summ2-8, summ2-8 mkk1/2, mkk6-2, mkk6-3,  pad4-1and the CA-MPK4 transgenic  line were described previously (Glazebrook, Rogers et al. 1996, Ichimura, Casais et al. 2006, Nakagami, Soukupova et al. 2006, Gao, Liu et al. 2008, Takahashi, Soyano et al. 2010, Berriri, Garcia et al. 2012, Zhang, Wu et al. 2012). The summ4-1D single mutant was isolated through backcrossing the triple mutant summ4-1D mkk1/2 to wild type Col-0 plants. The summ4-1D mekk1-1 double mutant was obtained by crossing summ4-1D mkk1/2 with mekk1-1. The summ4-1D mpk4-3 double mutant was obtained by crossing summ4-1D mkk1/2 with mpk4-3. The anp2-2 anp3-3 double mutant was obtained by crossing anp2-2 (Salk_144973) and anp3-3 (Salk_081990) obtained from the Arabidopsis Biological Resource Center. The anp2-2 anp3-3 CA-MPK4, anp2-2 anp3-3 summ2-8 and anp2-2 anp3-3 pad4-1 triple mutants were obtained by crossing anp2-2 anp3-3 with CA-MPK4CA, summ2-8 and pad4-1, respectively. Plants were grown at 23oC under 16 hr light/8 hr dark on soil or ½ Murashige and Skoog (MS) media. 2.5.2 Mutant characterization To determine gene expression levels, RNA was extracted from two-week-old seedlings grown on ½ MS media using EZ-10 Spin Column Plant RNA Mini-Preps Kit (Bio Basic, Canada). Genomic DNA contamination was removed by treatment with RQ1 RNase-Free DNase (Promega). Reverse Transcription was carried out using M-MuLV reverse transcriptase (New England Biolabs). Real-time PCR was performed using SYBR Premix Ex Taq II (Takara). Each experiment was repeated with three independent RNA samples. Primers of PR1, PR2 and ACTIN1 used for  40  RT-PCR were previously described (Sun et al., 2015). Primers used for MKK6 expression are listed in Supplemental Table 1. H. a. Noco2 infection was performed on two-week-old seedlings. The seedlings were sprayed with spore suspensions at a concentration of 50 000 spores per ml water. The plants were covered with a clear dome and kept at 18°C under 12 h light/12 h dark cycles in a growth chamber. Samples were collected 7 days later and spores on the plants were resuspended in water and counted using a hemocytometer. Infection results were scored as previously described (Bi, Cheng et al. 2010). P.syringae pv tomato DC3000 hrcC- infection was performed on four-week-old plants grown in short-day conditions. Two leaves of each plant were infiltrated with Pto DC3000 hrcC- (OD600=0.002) and one leaf disc from each infiltrated leaf was collected. The two leaf discs from the same plants were mixed together and regarded as one sample. These samples were grinded and diluted, then plated on LB plates. Colony-forming units (CFU) were calculated by counting colonies. 2.5.3 Map-based cloning of SUMM4 For crude mapping of summ4-1D, the summ4-1D mkk1/2 triple mutant was crossed with Landsberg erecta (Ler). F2 plants homozygous for mkk1/2 were selected for linkage analysis. summ4-1D mkk1/2 was also crossed with wild type Col-0 plants to obtain the summ4-1D single mutant. Plants homozygous for mkk1/2 and heterozygous for summ4-1D were also identified in the F2 generation and their progeny were used for fine mapping of summ4-1D. Markers for fine mapping were designed based on single nucleotide polymorphisms (SNPs) identified by sequencing the genome of summ4-1D mkk1/2 using Illumina sequencing. All primer sequences are listed in Supplemental Table 1. For testing whether the summ4-1D mutation is responsible for the suppression of the mkk1/2 mutant phenotype, the SUMM4 gene including the  41  mutation in the promoter region was amplified from the genomic DNA of summ4-1D mkk1/2 by PCR using primers MKK6-BamHI-F and MKK6-PstI-R. The DNA fragment was cloned into a modified pCambia1305 vector to express MKK6 under the mutant version of its native promoter. The construct was transformed into plants homozygous for mkk1 and heterozygous for mkk2 by the floral dipping method (Clough and Bent 1998). Transgenic plants homozygous for mkk1 mkk2 were identified by PCR in the T1 generation. 2.5.4 Split luciferase assay For testing interactions between MKK6 and MEKK1 or MPK4, cDNA of MKK6 was amplified by PCR using primers MKK6-cLuc-F and MKK6-cLuc-R and cloned into pCamiba 1300 CLuc (Chen, Zou et al. 2008) to express MKK6CLuc under a 35S promoter. cDNA fragments of MEKK1 and MPK4 were excised from pMEKK1-YCE and pMPK4-YCE  (Gao, Liu et al. 2008) and cloned into pCamiba 1300 NLuc (Chen, Zou et al. 2008) to express MEKK1NLuc and MPK4NLuc under a 35S promoter. 30-day-old tobacco leaves were infiltrated with Agrobacteria (OD600 = 0.2) carrying constructs expressing MKK6CLuc and MEKK1NLuc or MKK6CLuc and MPK4NLuc, along with the negative controls. Luciferase activity was measured using a plate reader. Plants were kept at 23oC under 16 hr light/8 hr dark condition for 2 days before assaying for luciferase activities.    42  Chapter 3 Future directions and conclusions  MAPK cascades play important roles in regulating a variety of biological processes in plants. The ANPs-MKK6-MPK4 cascade was originally proposed to regulate cytokinesis in Arabidopsis. My master thesis work suggested that this cascade also plays an important role in regulation of defense responses mediated by PAD4.  Defense responses in anp2 anp3 and mkk6 are only partially suppressed by introducing the constitutively active variant of MPK4. It’s possible that the MPK4 activity in CA MPK4 is lower than that activated by its upstream MAP2K, thus the defense responses in anp2 anp3 and mkk6 cannot be completely inhibited by CA MPK4. Alternatively, there could be other MAP kinases that function redundantly with MPK4 downstream of ANP2/ANP3 and MKK6.  Previous study showed that ANP3 can interact and phosphorylate MKK6 (Takahashi, Soyano et al. 2010, Zeng, Chen et al. 2011). To further confirm that MKK6 functions downstream of ANP2/ANP3 in plant immunity, a constitutively activate form of MKK6 will be transformed into anp2 anp3 to test it can suppress defense responses in anp2 anp3 plants.  PAD4 was found to function downstream of ANP2/ANP3, suggesting that one or more TIR-NB-LRR R proteins could function downstream of the ANP2/ANP3-MKK6-MPK4 cascade. To identify signaling components required for constitutive defense responses in anp2 anp3, a suppressor screen of anp2 anp3 has been carried out. A number of putative suppressors with wild-type morphology were isolated. Further characterization of these suppressor mutants and isolation of the mutant gene by positional cloning will help us better understand the function of the ANPs-MKK6-MPK4 cascade in plant immunity.    43  References Asai, T., G. Tena, J. Plotnikova, M. R. 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