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Towards comprehensive understanding of PLEIOTROPIC REGULATORY LOCUS 1 associated resistance signalling Weihmann, Tabea 2012

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Towards comprehensive understanding of PLEIOTROPIC REGULATORY LOCUS 1 associated resistance signalling  by  Tabea Weihmann Dipl. Biol., Rheinisch-Westfaelische Technische Hochschule Aachen, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies (Botany)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) March 2012 © Tabea Weihmann, 2012  Abstract Plants employ a multi-layered protection system to recognize pathogen presence and act upon intrusion. The conserved MOS4-associated complex (MAC) participates in the triggered signal transduction relay and contributes to the build-up of sound resistance. PLEIOTROPIC REGULATORY LOCUS 1 (PRL1), a MAC component with predicted structural function, is needed for a healthy immune response. Loss of this WD40 protein results in substantially higher pathogen colonization in Arabidopsis mutants. To dissect signalling steps downstream of the MAC, a mutant allele of PRL1 was chosen as the basis for a genetic suppressor screen. From this screen, both dominant and recessive mutants with defects in candidate genes were isolated, and two suppressors were cloned using map-based cloning techniques.  Characterization of the first dominant mutant revealed a gain-of-function mutation in PRL2, the homolog of PRL1. Although similar in sequence, the expression of PRL2 is greatly reduced in wild-type plants and functional analysis had not been attempted. Using the dominant prl2-1d allele and complementary mutants, full functional equivalence between the related proteins was established by means of defence –testing assays and evaluation of morphological criteria. This investigation revealed unequal genetic redundancy between the homologs; PRL2 has retained residual but relevant expression levels compared to the higher expressed PRL1. PRL2 also displays modified expression patterns, potentially indicative of developing tissue specificity.  The haplo-insufficient SUPPRESSOR OF prl1, 2 (SOP2) gene is an intriguing discovery in PRL1 signal relay. Devoid of known sequence motifs, SOP2 encodes a novel nuclear protein with homologs limited to the plant kingdom. Several lines of evidence support a dosagedependent mechanism, mediated by SOP2, which is prone to interference by a spoiler protein. Both the obtained dominant-negative sop2-1D allele and a recessive sop2 mutation fully suppress prl1-related phenotypes, however neither one causes impaired resistance in single mutant analysis. Although specifics of SOP2 functionality in the context of plant resistance signalling remain to be fully resolved, clues from epistasis analysis point towards a PRL1 centered relationship and do not support SOP2 as a target of the MAC. ii  Preface Some of the work presented in this thesis has been a collaborative effort. Contributions made by the candidate and fellow scientists are outlined below.  Chapter 1: The candidate wrote the chapter. The figure used in this chapter has been designed by the candidate and has been published in the chapter “Plant Innate Immunity” of Plant – Environment Interactions, Signalling and Communication in Plants, Springer, 2009. Editorial support was provided by Dr. Li, Dr. Kronstad and Dr. Samuels.  Chapter 2: The candidate wrote the chapter. A version of this chapter will be included in a manuscript tentatively titled “ A gain-of-function mutation in PLEIOTROPIC REGULATORY LOCUS 2 reveals unequal genetic redundancy and functional equivalence with PRL1”, which is in preparation for publication. Anticipated authors list: Weihmann, T., Palma, K. and Li, X.  Experiments were co-designed by the candidate and Dr. Li and carried out by the candidate. Dr. Palma (BC Cancer Research, Vancouver) carried out the first stages of the prl1 suppressor screen until screening of M2 mutants. Dr. Yuelin Zhang (NIBS, P.R. China) provided all three JAtY clones and Shuxin Li, from Dr. Zhang’s laboratory, obtained ratios of segregating F2 progeny used in Table 1. Angela Chiang (Bohlmann Lab, UBC) assisted in real-time PCRanalysis and James Robertson, MSc. provided PAC polymerase used in prl2-1D cloning approaches. Dr. Li supervised the work and manuscript preparation and provided editorial support. Dr. Kronstad and Dr. Samuals also provided editorial support.  Chapter 3: The candidate wrote the chapter. A version of this chapter tentatively titled “ A gainof-function mutation in PLEIOTROPIC REGULATORY LOCUS 2 reveals unequal genetic  iii  redundancy and functional equivalence with PRL1”, is in preparation for publication. Anticipated authors list: Weihmann, T., Palma, K. and Li, X.  Experiments were co-designed by the candidate and Dr. Li and carried out by the candidate. Dr. Palma (BC Cancer Research, Vancouver) isolated the prl1-2 prl2-2 double mutant. Shuxin Li (Zhang laboratory, NIBS, P.R. China) assisted in mapping prl2-1D. Dr. Li supervised the work and manuscript preparation and provided editorial support. Dr. Kronstad and Dr. Samuels also provided editorial support.  Chapter 4: The candidate wrote the chapter. A version of this chapter tentatively titled “ The plant specific SOP2 functions in signal relay mediated by the evolutionary conserved WD40 protein PRL1” is in preparation for publication. Anticipated authors list: Weihmann, T., Xia, S., Lee, E., Sack, F. and Li, X.  Experiments were co-designed by the candidate and Dr. Li and carried out by the candidate. Dr. EunKyoung Lee (Sack laboratory, UBC) performed fluorescence microscopy for localization of SOP2 shown in Figure 15. Shuxin Li (Zhang laboratory, NIBS, P.R. China) assisted in mapping sop2-1D. Jaclyn Dee, MSc. (Berbee laboratory, UBC) assisted in preparing phylogenetic relationships shown in Figure 12. Dr. Li supervised the work and manuscript preparation and provided editorial support. Dr. Kronstad and Dr. Samuels also provided editorial support.  Chapter 5: The candidate wrote the chapter. Editorial support was provided by Dr. Li, Dr. Samuels and Dr. Kronstad.  iv  Table of Contents Abstract................................................................................................................................. ii Preface ................................................................................................................................. iii Table of Contents ................................................................................................................. v List of Tables ....................................................................................................................... ix List of Figures ....................................................................................................................... x List of Abbreviations ........................................................................................................... xi Acknowledgments ............................................................................................................. xiii Dedication .......................................................................................................................... xiv 1 Introduction ....................................................................................................................... 1 1.1  Inducible plant defence systems .................................................................................. 1  1.2  Pathogen perception and signalling induced by cell surface receptors ......................... 1  1.3  Virulence factors of pathogenic microbes ..................................................................... 4  1.4  Intracellular receptors initiate effector-triggered defence .............................................. 7  1.5  Biotrophic pathogens induce salicylic acid-dependent signalling ................................ 10  1.6  Modifier of snc1 genes and the MOS4-associated complex ....................................... 14  1.7  PLEIOTROPIC REGULATORY LOCUS 1 (PRL1) ..................................................... 16  1.8  Research objectives................................................................................................... 18  1.8.1  Compilation and preliminary analysis of suppressor of prl1 (sop) mutants .................. 19  1.8.2  Analysis of the relationships between PRL1 and homologous gene, PRL2 .................. 20  1.8.3  Characterization of SOP2, member of a novel gene family with unknown function ..... 20  v  2 A prl1 suppressor screen yielded twenty-two mutants with defects in candidate genes ............................................................................................................................... 23 2.1  Introduction ................................................................................................................ 23  2.2  Material and methods ................................................................................................ 24  2.2.1  Plant material and growth ................................................................................ 24  2.2.2  Agrobacterium tumefaciens mediated mutagenesis ........................................ 25  2.2.3  EMS mutagenesis and primary morphological screen ..................................... 25  2.2.4  Secondary resistance screen .......................................................................... 26  2.2.5  Assessment of genetic inheritance .................................................................. 26  2.3  Results....................................................................................................................... 27  2.3.1  Complete and partial suppression of prl1 phenotypes by sop mutations.......... 27  2.3.2  Restored R protein mediated resistance in most prl1-2 sop mutants ............... 27  2.3.3  Suppressor phenotypes are caused by dominant, semi-dominant and recessive mutations ........................................................................................................ 28  2.4  Discussion ................................................................................................................. 28  3 A gain-of-function mutation in PLEIOTROPIC REGULATORY LOCUS 2 reveals unequal redundancy and functional equivalence between this WD40 protein and the close homolog PRL1 ....................................................................................................... 33 3.1  Introduction ................................................................................................................ 33  3.2  Materials and methods ............................................................................................... 35  3.2.1  Plant material and growth ................................................................................ 35  3.2.2  Morphological characterization ........................................................................ 35  3.2.3  Infection assays .............................................................................................. 36  3.2.4  Positional cloning ............................................................................................ 36 vi  3.2.5  Quantification of PRL2 mRNA levels ............................................................... 37  3.2.6  Single and double mutant construction ............................................................ 37  3.2.7  Transformation of JAtY clones......................................................................... 38  3.3  Results....................................................................................................................... 38  3.3.1  sop1-1D fully suppresses prl1-associated phenotypes .................................... 38  3.3.2  Map-based cloning of sop1-1D identifies a molecular lesion in PLEIOTROPIC REGULATORY LOCUS 2 (PRL2) ................................................................... 39  3.3.3  sop1-1D is a gain-of-function allele of PRL2 .................................................... 41  3.3.4  PRL2 and PRL1 exhibit high structural and sequence homology ..................... 42  3.3.5  Unequal genetic redundancy between PRL1 and PRL2 .................................. 43  3.3.6  prl1-2 prl2-2 mutants are impaired in basal and R-protein mediated ................... resistance ........................................................................................................ 44  3.3.7 3.4  prl2-1D single mutants do not show enhanced resistance to pathogens .......... 45  Discussion ................................................................................................................. 46  4 A dominant mutation in an uncharacterized gene identifies a component of PRL1 signalling specific to the plant kingdom ....................................................................... 57 4.1  Introduction ................................................................................................................ 57  4.2  Material and methods ................................................................................................ 59  4.2.1  Plant material and growth ................................................................................ 59  4.2.2  Plant pathogens and infection assays ............................................................. 60  4.2.3  Positional cloning ............................................................................................ 61  4.2.4  Single and double mutant construction ............................................................ 61  4.2.5  Phylogenetic analysis ...................................................................................... 62  4.2.6  Localisation of SOP2-GFP .............................................................................. 62  4.2.7  Quantification of SOP2 mRNA levels............................................................... 63 vii  4.3  Results....................................................................................................................... 63  4.3.1  Dominant suppression of prl1 loss-of-function phenotypes by sop2-1D ........... 63  4.3.2  A leucine-for-proline substitution in an uncharacterized protein causes differential interference of prl1-related signalling.............................................. 64  4.3.3  SOP2 and SOP2h are plant-specific proteins .................................................. 66  4.3.4  SOP2 and SOP2h are not essential for plant defence ..................................... 66  4.3.5  Dosage-dependent suppression of prl1-2 by a loss-of-function mutation in SOP2 supports dominant-negative activity of sop2-1D .................................... 67  4.3.6  SOP2 gene expression is only slightly altered in MAC mutants ....................... 68  4.3.7  SOP2 is a nuclear protein................................................................................ 69  4.3.8  sop2-2 does not suppress MAC mutations atcdc5-2 or mos4-1 ....................... 70  4.4  Discussion ................................................................................................................. 70  5 Concluding discussion and outlook .............................................................................. 83 5.1  Suppressors of prl1 signalling .................................................................................... 83  5.2  Analysis of PRL2........................................................................................................ 85  5.3  Discovery of SOP2..................................................................................................... 87  References .......................................................................................................................... 91 Appendices ....................................................................................................................... 112 Appendix 1: Arabidopsis transformation protocol ............................................................ 112 Appendix 2: Molecular marker......................................................................................... 113 Appendix 3: Gene expression profiles of PRL1 and PRL2 .............................................. 114 Appendix 4: Gene ID’s of SOP2 homologs...................................................................... 115 Appendix 5: Gene expression profile of SOP2 ................................................................ 116  viii  List of Tables Table 1. Analysis of sop mutants ...................................................................................................... 31 Table 2. sop mutants exhibit complete and partial suppression of prl1 phenotypes .................... 32  ix  List of Figures Figure 1. Signalling events involved in plant innate immunity ........................................................ 22 Figure 2. sop1-1D suppresses prl1 related phenotypes ................................................................. 51 Figure 3. Map-based cloning of sop1-1D ......................................................................................... 52 Figure 4. Overexpression of PRL2 is able to complement prl1 mutant defects............................ 53 Figure 5. Alignment of PRL1 and PRL2 protein sequences ........................................................... 54 Figure 6. Enhanced phenotypes in prl1 prl2 double mutants compared to single mutants ......... 55 Figure 7. Basal and R protein mediated resistance is unaffected in prl2-1D ................................ 56 Figure 8. prl1 phenotypes are suppressed in prl1-2 sop2-1D ........................................................ 75 Figure 9. Map-based cloning of sop2-1D ......................................................................................... 76 Figure 10. SOP2 and SOP2h are homologous proteins ................................................................. 77 Figure 11. Phylogenetic analysis of SOP2 homologs in plants ...................................................... 78 Figure 12. Mutations in SOP2 and SOP2h do not cause morphological or disease-related phenotypes ......................................................................................................................................... 79 Figure 13. sop2-2 is a semi-dominant suppressor of prl1-phenotypes ......................................... 80 Figure 14. SOP2 localizes to the nucleus ........................................................................................ 81 Figure 15. sop2-2 does not affect mutant phenotypes of atcdc5-2 or mos4-1 ............................. 82  x  List of Abbreviations AtCDC5: Arabidopsis thaliana CELL DIVISION CYCLE 5 Avr: Avirulence BAK1: BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 BIK1: BOTRYTIS-INDUCED KINASE 1 CC: coiled-coil cfu: colony forming units Col: Arabidopsis thaliana ecotype Columbia DCX: DDB1/CULLEN4/protein X DWD: DDB1 binding WD40 EDS1: ENHANCED DISEASE SUSCEPTIBILITY 1 EFR: EF-TU RECEPTOR EMS: ethyl methanesulfonate ETI: effector-triggered immunity FLS2: FLAGELLIN SENSING 2 F1: first filial generation F2: second filial generation GFP: GREEN FLUORESCENT PROTEIN H.a.: Hyaloperonospora arabidopsidis InDel: Insertion/Deletion kD: kilodalton LB: luria bertani Ler: Arabidopsis thaliana ecotype Landsberg MAC: MOS4-associated complex MAPK: MITOGEN ACTIVATED PROTEIN KINASE MOS: MODIFIER OF SNC1 MS: murashige-skoog NB-LRR: mucleotide binding – leucine rich repeat NCBI: National Centre for Biotechnology Information NLS: nuclear localisation signal NPR1: NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 xi  OD: optical density PAD4: PHYTOALEXIN DEFICIENT 4 PAMP: pathogen associated molecular pattern PCR: polymerase chain reaction PR: PATHOGENESIS RELATED PRL1: PLEIOTROPIC REGULATORY LOCUS 1 PRL2: PLEIOTROPIC REGULATORY LOCUS 2 P.s.m.: Pseudomonas syringae pv maculicola P.s.t.: Pseudomonas syringae pv tomato PTI: PAMP-triggered immunity RPP4: R protein: Resistance protein SA: salicylic acid snc1: suppressor of npr1, constitutive 1 SNP: single nucleotide polymorphism SOP: SUPPRESSOR OF PRL1 SOP2h: SOP2 homolog T-DNA: transfer DNA T1: first transgenic generation T2: second transgenic generation TIR: Toll/Interleukin-1 TTSS: type three secretion system WD40: Trp-Asp (W-D) WRKY: Trp-Arg-Lys-Tyr (W-R-K-Y) Ws: Arabidopsis thaliana ecotype Wssilewskija  xii  Acknowledgments I would like to thank Prof. Xin Li for the opportunity to carry out PhD studies in her lab and for the guidance and support that I have received throughout these five years. I am also grateful for advice and support, whenever I needed it, from my committee members Prof. Jim Kronstad, Prof. Lacey Samuels and Prof. Fred Sack. My thanks goes to our collaborators, who have given their time and provided expertise, tools and reagents: Dr. Kris Palma, Dr. Yuelin Zhang, Dr. EunKyoung Lee, Dr. Shitou Xia, Jaclyn Dee and Shuxin Li. Interactions with colleagues are important aspects of working in a lab and I feel truly blessed to have met so many extraordinary people. Conversations, may it be scientifically or otherwise, with Jacqui, Hugo, Sandra, Marcel, YuTi, Fang, Patrick, Yan, Yuxiang, Jin and Oliver have reshaped my view of the world. I really hope that the newer students in the lab, Shuai, Kaeli, Virginia, Xuejin, and Xuezi will experience a similar community-focused work environment…it makes all the difference when things get rough. A special thanks goes to our student workers, especially Emma, Aliya, Jenny and Larissa, who kept the lab in a running order despite the never ending stream of dirty flats and glassware that had to be washed, and who could be trusted with all the little but important jobs. The administrative staff at MSL and in the Botany department is amazing: I am indebted to Victor, who can fix anything and to Stephen and Vince, who had to deal with numerous computer related emergencies over the years. Veronica Oxtoby and Sarah Ruddick solved many minor and major problems of mine and my thank you also goes to Alexandra Kielb, Gerado Andres and Pal Bains. I have made friends in Vancouver and it is because of this supportive network that my Grad life stayed positive. Here is to memories of fantastic parties, weekends away, living with Ph.D. flatmates, snowboarding, sailing, dinner club, games nights or just hanging out, debating the problems of the world…you guys know who you are . My partner Andrew, my family in Germany and South Africa and my friends back home have been vital to the success of this Ph.D. When I set out to Canada in 2005, armed with a bicycle and a backpack, even I did not anticipate the magnitude of this adventure. Every single one of you has since played a part in this endeavour and your loyalty, friendship and support humbles me. I am so glad that an auntie living in Canada (!) makes up for some of my shortcomings in visiting ability but I miss you terribly, Esther. And above all, this is for you, Boyfriend. xiii  Dedication  To my family and friends in Canada, Germany and South Africa, who are unwavering in their support for my endeavours.  xiv  1  Introduction  1.1  Inducible plant defence systems  Plants are constantly exposed to a wealth of microbes which seek access to nutrients. Fortunately, inducible layers of defence are capable of conveying sound resistance towards many microbes thus rendering disease an exception. Following detection of conserved pathogen-associated molecular patterns (PAMPs) by immunity receptors located on the cell surface, broad range protection is activated and only specialized pathogens can overcome these defences. Innovative pathogens have found ways to evade or suppress such early resistance mechanisms through the use of virulence factors which are delivered into a host cell. Intracellular receptors, denoted resistance proteins (R proteins), detect the presence or activity of these pathogen-delivered molecules and induce strong immunity responses often leading to localized cell death and halting of pathogen invasion. PAMP and effector triggered immunity signalling is complex. Both proteins and small molecules with diverse functions are utilised and also involves substantial redistribution of cellular resources and efforts. Whereas infections by avirulent pathogens are halted by the combined defence layers, virulent pathogens defy these efforts and disease ensues.  1.2  Pathogen perception and signalling induced by cell surface  receptors Plants are equipped with a receptor-based surveillance system oriented towards the extracellular environment which recognizes components central to microbial life. Fungal chitin, bacterial flagellin, lipopolysaccharides or the cellulose binding elicitor lectin (CBEL) from Phytophthora parasitica, a filamentous oomycete, are PAMPs that are recognized by 1  specialized pattern receptors (Fig. 1) (Gust et al., 2007; Newman et al., 2007; Gaulin et al., 2006).  Closure of stomata contributes to the plants immune response and is in part mediated by the detection of microbial flagellin and lipopolysaccharides on the plant surface, in an effort to restrict bacterial invasion (Zhang et al., 2008; Melotto et al., 2006).To counteract this, the bacterial strain Pseudomonas syringae pv tomato (P.s.t) DC3000 releases the phytotoxin coronatine which induces reopening of stomata and is critical for overall virulence (Zeng and He, 2010; Melotto et al., 2006).  Recognition of fungal microbes through chitin detection is mediated by CERK1 (CHITIN ELICITOR RECEPTOR KINASE 1), a LysM domain receptor kinase (Iizasa et al., 2010; Petutschnig et al., 2010; Miya et al., 2007). CERK1, and two other LysM domain proteins LYM1 (LysM DOMAIN PROTEIN 1) and LYM3, also participate in peptidoglycan sensing and thus immunity to bacterial pathogens (Willmann et al., 2011). The perception of bacterial flagellin is probably the best understood mechanism involving a PAMP molecule and its respective receptor: in this interaction, the leucine-rich repeat receptor kinases (LRR-RK) FLAGELLIN SENSING2 (FLS2) recognizes the flg22 epitope of flagellin and subsequently interacts with several other LRR-RK’s, among them BAK1/SERK3 (BRASSINOSTEROID INSENSITIVE1ASSOCIATED KINASE1/ SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE3), SERK1 and BKK1/SERK4 (BAK1-LIKE1) (Roux et al., 2011; Chinchilla et al., 2007; Heese et al., 2007).  The cytoplasmic kinase BIK1 (BOTRYTIS-INDUCED KINASE 1), originally discovered in the immunity response against necrotrophic fungal pathogens, also associates with FLS2-BAK1 and may be the first to be phosphorylated in a sequential series of reciprocal trans phosphorylation steps between kinases (Lu et al., 2010b; Wang et al., 2008). BAK1 might also differentially phosphorylate other complex members, leading to varying signal outputs 2  (Schwessinger et al., 2011). PAMP signalling might be further propagated through release of BIK1 from the receptor complex (Lu et al., 2010b).  To fine tune responses, activated FLS2 receptors are degraded following ligand-induced endocytosis (Robatzek et al., 2006). Ubiquitination of FLS2 by two BAK1 associated U-box E3 ligases, PUB12 and PUB13, suggests degradation via the plants proteasome machinery (Lu et al., 2011). Recently, a role of the gaseous phytohormone ethylene in regulation of FLS2 has been demonstrated. Presence of ETHYLENE-INSENSITIVE 2 (EIN2) and endogenous levels of ethylene result in binding of the transcription factor EIN2 to the FLS2 promoter sequence, possibly replenishing receptor numbers in a positive feedback loop (Boutrot et al., 2010).  There is growing evidence that BAK1 forms similar ligand-induced complexes with other receptors including PEPR1( PEP RECEPTOR1) and PEPR2, which recognize the PAMP and wound-induced endogenous molecule Pep1, and EFR (EF-TU RECEPTOR), the receptor recognizing the bacterial elongation factor Tu (Roux et al., 2011; Krol et al., 2010; Postel et al., 2010; Zipfel et al., 2006). Mutants of pattern recognition receptors usually show heightened susceptibility towards pathogens underlining the importance of PAMP-triggered immunity (PTI) in the overall defence response (Nicaise et al., 2009).  Following PAMP perception, a cascade of mitogen activated protein kinases (MAPK) is induced, likely including sequential phosphorylation of at least a MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK) and a MAPK. Using a protoplast system to identify MAPK’s in flg22-activated PTI outputs, signal routing through MEKK1, MKK4/5 and MPK 3/6 was initially suggested (Asai et al., 2002). Subsequent work, however, suggests that MEKK1 instead functions in a cascade with MKK1/2 and MPK4, which negatively regulates resistance (Gao et al., 2008; SuarezRodriguez et al., 2007; Ichimura et al., 2006). Interaction of MEKK1 with MKK1/2 mainly occurs at the plasma membrane, whereas MPK4 and MKK2 have been shown to associate both on the 3  membrane surface and in the nucleus, suggesting that signal relay might function through relocation of MKK1 and MKK2 (Gao et al., 2008). It thus appears that FLS2 activates an unknown MAPKKK followed by phosphorylation of MKK4/5 and MPK3/4 which positively regulate PTI but also triggers a MAPK cascade with inhibitory influence on immunity (Fig. 1) (Rodriguez et al., 2010) . A negative defence response modulated by an MPK4 homolog was also monitored in soybean (Liu et al., 2011).  Both antagonistic pathways are expected to affect defence gene expression through WRKYtype transcription factors such as WRKY22 and WRKY29, which probably function downstream of MPK3/6 (Asai et al., 2002). The MPK4 substrate MKS1, which is required for resistance in mpk4 mutants, has been recently shown to interact with both WRKY33 and MPK4 using the Nterminal domain (Petersen et al., 2010; Andreasson et al., 2005). A complex consisting of MPK4, WRKY33 and MKS1 appears to be activated through defence signalling, leading to phosphorylation of MKS1 and release of the transcription factor. WRKY33 is later recruited to the promoter of PHYTOALEXIN DEFICIENT 3, a gene required for synthesis of phytoalexin camalexin (Qiu et al., 2008). Phosphatases also contribute to the coordination of defence responses through de-phosphorylation of kinases. MAPK phosphatase 2 interacts with both MPK3 and MPK6, likely influencing signalling capacities (Lumbreras et al., 2010).  1.3  Virulence factors of pathogenic microbes  Due in large parts to broad range PAMP-signalling, plants fight off pathogenic take-over attempts in most cases. The mechanisms resulting in PTI are consequently the main targets of pathogen-delivered virulence factors, called effectors. An essential factor in bacterial virulence, the type three secretion system (TTSS), is encoded by the hrp/hrc (hypersensitivity response and pathogenicity/hrp conserved) gene cluster which assembles into a multiprotein structure capable of translocating molecules into the host cytoplasm (Fig.1) (Tampakaki et al., 2010). 4  The inventory of effectors thought to be injected by Pseudomonas syringae pv phaseolicola 1448a amounts to 27 molecules and which is similar in scale to the predicted 28 effector repertoire of P.s.t.DC3000 (Zumaquero et al., 2010; Cunnac et al., 2009). Between 30-40 molecules are translocated by plant pathogenic Xanthomonas bacteria (Buttner and Bonas, 2010).  The virulence promoting function of many effectors remains elusive although progress has been made, e.g. early targets of effectors are pattern recognition receptors. Binding of P.s. effector AvrPto to FLS2 and EFR in Arabidopsis and to LeFLS2 in tomato has been previously shown to correlate with hinderance of PTI, possibly through inhibiting of kinase signalling ability (Xiang et al., 2008). Phosphorylation of BIK1, the proposed first target of FLS2 is blocked in the presence of AvrPto (Xiang et al., 2011).  The sequence-distinct bacterial effector avrPtoB mimics the structure and function of an eukaryotic E3 ligase to mediate ubiquitination and subsequent degradation of host PTI kinases in susceptible plants (Ntoukakis et al., 2009; Rosebrock et al., 2007; Janjusevic et al., 2006). Targets of AvrPtoB, which is widely found among Erwinia, Xanthomonas and Pseudomonas strains, include the tomato kinase Fen as well as CERK1, the receptor kinase responding to both chitin and peptidoglycan patterns (Willmann et al., 2011; Gimenez-Ibanez et al., 2009). In vitro kinase activity of Bti9, the most sequence similar LysM receptor-like kinase to CERK1 in tomato, is also halted in the presence of AvrPtoB (Zeng et al., 2011). AvrPto and AvrPtoB may also bind the regulatory protein BAK1, potentially interfering with signal relay mediated by multiple receptors (Lu et al., 2010a; Shan et al., 2008).  Another example of an effector hijacking host mechanisms is the TAL family of transcription activator-like molecules used by Xanthomonas spp. and Ralstonia solanacearum. TAL effectors imitate eukaryotic transcription factors and specifically initiate gene expression upon binding to 5  the UPT (UPREGULATED BY TAL EFFECTORS) box of host target genes (Boch et al., 2009; Moscou and Bogdanove, 2009).  Delivery and virulence functions of oomycete and fungal effectors are less well understood. An uptake signal motif with the consensus sequence RXLR (arginine, “any amino acid”, leucine, arginine), is shared by many oomycete effectors and is important for translocation into the host plant (Dou et al., 2008; Whisson et al., 2007). One possible mechanism involves binding of the RXLR motif to phosphatidyl inositol phosphatases (PIP) on the outer surface of the plant plasma membrane, followed by lipid raft-mediated endocytosis of the complex (Kale et al., 2010). Requirement of the RXLR-PIP interaction for translocation is currently debated (Yaeno et al., 2011).  Avr3b, an RXLR effector from Phytophthora sojae, is an ADP-ribose/NADH pyrophosphorylase (Dong et al., 2011). In plants, some proteins with a similar Nudix motif have been shown to act as negative regulators of resistance, i.e. AtNUDT7 (ARABIDOPSIS THALIANA NUDIX HYDROLASE HOMOLOG 7) (Bartsch et al., 2006b). Defects in AtNUDT7 result in altered cellular redox levels, suggesting a role for this protein and similarly, Avr3b, in early defence modulation (Ge et al., 2007).  Phytophthora infestans encodes two forms of the RXLR effector Avr3a which suppress PAMPlike elicitin INF1 induced programmed cell death (Bos et al., 2009). INF1 interacts with CMPG1, a host E3 ubiquitin ligase which in turn is stabilised by Avr3a (Gonzalez-Lamothe et al., 2006). Although targets of CMPG1 activity are unknown, maintaining processes is beneficial to the pathogen. Silencing of the Avr3a genes results in significantly reduced virulence, suggesting these factors are required for full pathogenicity (Bos et al., 2010).  6  Functional characterization of individual effectors has proven difficult since mutating one virulence factor rarely reduces virulence, likely due to redundancy between pathogen-delivered molecules. Creation and comprehensive analysis of pathogenic bacterial strains with double and triple effector mutations may overcome some of the problems (Zumaquero et al., 2010). A TTSS based system in P.s.t. DC3000-LUX was recently used to investigate oomycete effectors and may also facilitate accelerated screening procedures for this type of pathogen (Fabro et al., 2011)  1.4  Intracellular receptors initiate effector-triggered defence  Despite the amount and functional diversity of virulence factors, the plant immune system often succeeds in limiting pathogen proliferation. A second layer of inducible defences is mediated by the products of resistance (R) genes which detect the presence or activities of effectors, also called Avirulence (Avr) proteins. In many cases, such activity is an attempt by the pathogen to interfere with PTI responses. R genes encode intracellular receptors that initiate effectortriggered immunity (ETI) after perception of a Avr-associated danger cues (Fig. 1) (Jones and Dangl, 2006).  Plant immunity receptors are encoded by at least five different classes of resistance-associated genes (Glowacki et al., 2011; Dangl and Jones, 2001). PAMP receptors usually reside in plasma membranes whereas the largest class of ETI receptors localize to the cytosol. Recent genome-wide predictions suggest a large number of proteins, between 159 – 174, as members of the nucleotide binding – leucine rich repeat (NB-LRR) family of R proteins (Guo et al., 2011; Chen et al., 2010). NB-LRR’s are further subdivided into two categories depending on either homology of their N-terminal domain with Drosophila Toll and human interleukin-1 proteins (TIR) or according to an N-terminal coiled coil motif (CC). RPM1 (RESISTANCE TO PSEUDOMONAS SYRINGAE PV MACULICOLA 1) is a CC-NB-LRR class protein whereas 7  RPS4 (RESISTANCE TO PSEUDOMONAS SYRINGAE 4) and RPP1 (RESISTANCE TO PERONOSPORA PARASITICA 1) are examples of TIR-type R proteins (Takken and Tameling, 2009b).  In Arabidopsis, P.s. strains expressing the effector molecule AvrPphB elicit local cell death known as the hypersensitive response (HR), which halts the infection process. This process is dependent on the CC-NB-LRR R-protein RPS5, which is associated with the immediate target of the effector, the host kinase PBS1. Proteolytic cleavage of PBS1 by the effector protease AvrPphB presumably aids in bacterial colonization, however leads to ETI signalling and resistance in plants with the RPS5 gene (Shao et al., 2003). Cleavage of the host protein causes a modified phosphorylation state of the R protein and induces immunity responses (Ade et al., 2007).  The ETI response triggered by AvrPphB relies on AtMIN7 (ARABIDOPSIS THALIANA HOPM INTERACTOR 7), a guanine nucleotide-exchange factor targeted by the HopM1 effector (Nomura et al., 2011; Nomura et al., 2006). HopM1 destabilizes AtMIN7 levels in the plant via a 26S proteasome-dependent mechanism thus resulting in susceptibility towards P.s.pv.tomato. DC3000 (Nomura et al., 2006). In contrast, presence of avirulence proteins AvrPphB, AvrRpt2 or HopA1 blocks degradation. This suggests a mechanism in which earlier ETI responses triggered by avirulence proteins work to protect downstream targets of the signal transduction (Nomura et al., 2011). AtMIN7 and HopM1 are localized in the trans-Golgi/early endosome system, which supports increasing evidence towards a critical role of vesicle traffic in defence (Nomura et al., 2011; Frei dit Frey and Robatzek, 2009; Hoefle and Huckelhoven, 2008).  Activity associated with the previously introduced TAL effectors of Xanthomonas campestris pv. vesicatoria is recognized via an intriguing mechanism in pepper plants. Target genes of the TAL effector AvrBs3 include host genes with assumed beneficial function to the pathogen. But the 8  transcription factor- like effector will also bind a UPT box in the promoter region of the pepper resistance gene Bs3. Presence of Bs3 confers ETI signalling likely through induced synthesis of and subsequent signalling by Bs3, leading to resistance in plants (Romer et al., 2007). A similar mechanism might also mediate resistance of rice towards X. oryzae pv. oryzae. Effective defence in resistant plants is activated by the TAL effector AvrXa27, and is characterized through increased transcription of the rice R gene Xa27, which also carries an UPT box (Romer et al., 2009).  ETI receptors may also be directly interacting with Avr proteins, e.g. the TIR-NB-LRR RPP1 proteins present in Arabidopsis thaliana ecotypes co-purify with ATR1 (ARABIDOSIS THALIANA RECOGNIZED 1), an oomycete effector from several Hyaloperonospora arabidopsidis (H.a.) subspecies including EMOY, CALA and NOCO (Krasileva et al., 2010; Rehmany et al., 2005). Depending on recognition surface variants between RPP1-encoding ecotypes, interaction specifics may differ (Chou et al., 2011). Evidence further suggests that RPP1 variants associate with the effector in an inactive state. This would support the current hypthesis of an R protein activation switch, which possibly includes conformational changes (Krasileva et al., 2010; Lukasik and Takken, 2009).  Crystallization of both the CC domain of barley MLA10 and the TIR domain of flax L6 as protein dimers are consistent with activation models of R-proteins requiring conformational changes potentially through oligomerization. Site-directed mutagenesis of dimerization relevant residues resulted in the loss of signalling ability in planta, further supporting a role for multimeric receptor complexes at the beginning of ETI (Bernoux et al., 2011; Maekawa et al., 2011; Takken and Tameling, 2009a). Examples of proteins that aid in NB-LRR complex stabilization include RAR1, HSP90 and SGT1 (REQUIRED FOR MLA12 RESISTANCE, HEAT SHOCK PROTEIN 90, SUPPRESSOR OF THE G2 ALLELE OF SKP1 ) (Shirasu, 2009). Negative regulation of R-  9  protein signalling may be provided by the tetratrico peptide repeat protein SRFR1 (SUPPRESSOR OF rps4-RLD), which interacts with SGT1 in vivo (Li et al., 2010).  Indirect or direct association, as well as a general capability to recognize effector-induced modification of host proteins, are mechanisms predicted by the guard model of plant resistance (Dangl and Jones, 2001). However, in recent times, examples of effector recognition have been described that do not fit the hypothesized guarding mechanism of PTI-involved subjects. The AvrPto-Pto relationship is such an example. As described earlier, AvrPto interacts and inhibits PAMP receptors FLS2 and EFR; however, it also binds the tomato resistance protein Pto which closely resembles the structure of these receptor kinases and competes with FLS2 for interaction. An AvrPto-FLS2 complex promotes virulence, whereas interaction of AvrPto with Pto triggers strong defences and may suggest that the R-proteins’ main function is that of a decoy for AvrPto (Xiang et al., 2008). The two models differ solely in the proposed recognition mechanism of pathogenic activity. ETI is either triggered by an R protein after a guarded host protein has been compromised or through an alternative relationship between a decoy R gene and a virulence factor. The current understanding allows for both models (Block and Alfano, 2011; van der Hoorn and Kamoun, 2008).  1.5  Biotrophic pathogens induce salicylic acid-dependent signalling  PTI and ETI receptors both recognize ligands to activate defence and the induced signalling cascades use in some cases molecules common to both pathways. A major branch in plant immunity signalling is the salicylic acid (SA) signal route which is used by both PTI signal relay and ETI networks in response to (hemi) biotrophic pathogens such as H.a. ssp. and P.s. strains. In our current understanding, the lipase like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) is a primary component upstream of SA production and is required for activation of both TIR and CC-type R-protein signalling (Venugopal et al., 2009; Parker et al., 1996). EDS1 and 10  related protein PHYTOALEXIN DEFICIENT 4 (PAD4) localize to the cytosol as well as to the nucleus and are able to interact with each other (Wiermer et al., 2005; Feys et al., 2001). A third, similar protein, SENESCENCE ASSOCIATED GENE 101 (SAG101) is preferentially found in the nucleus where it forms a ternary complex which EDS1 and PAD4 (Zhu et al., 2011; Feys et al., 2005). Localisation of the components and also of the complex appear to be dependent on relative levels of binding partners; both SAG101 and PAD4 affect EDS1 distribution, however with antagonistic effects (Zhu et al., 2011). Such redistribution could be a mechanism to adjust defence outputs, as suggested by the need for balanced levels of EDS1 for fully functional resistance (Garcia et al., 2010).  The NDR1 (NONRACE-SPECIFIC DISEASE RESISTANCE) protein functions early in multiple CC-type R-protein signalling pathways and shows homology to integrins, a class of surface receptors that bind extracellular proteins and relate signals into the cell through connection to the cytoskeleton (Knepper et al., 2011; Gee et al., 2008; Century et al., 1995). Consequently, ndr1 mutants are compromised in the adhesion between the plasma membrane and the cell wall, and also show altered pathogen-induced electrolyte leakage, outlining the importance of maintaining cell integrity in plant resistance (Knepper et al., 2011)  Several genes important for SA production and accumulation have been previously identified. In response towards pathogen infection, SA responses are controlled by EDS1, PAD4 and Isochorismate synthase SID2/ICS1 (SA INDUCTION DEFICIENT2/ISOCHORISMATE SYNTHASE 1) which synthesises SA from the precursor chorismate (Wildermuth et al., 2001). ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) has homology to MATE transport family proteins, and may be involved in the transport of SA related molecules (Nawrath et al., 2002) The ALD1 protein (AGD2-LIKE DEFENCE RESPONSE PROTEIN 1) also contributes to the accumulation of SA and acts additively with PAD4 (Song et al., 2004).  11  In PTI responses, the association of calmodulin (CaM) with CBP60g, a member of the CaMbinding CBP60 family, positively affects SA accumulation either independent or potentially upstream of PAD4 (Wang et al., 2009). CaM binding is not required for the function of SARD1 (SAR DEFICIENT 1), a closely related and functionally redundant member of the CBP60 family of plant specific DNA binding proteins. In the presence of pathogens, both SARD1 and CBP60g are recruited to the promoter of SID2/ICS1 potentially affecting transcription of the SA-producing enzyme (Wang et al., 2011; Zhang et al., 2010).  PBS3 (AvrPphB SUSCEPTIBLE), a member of the GH3-like family of acyl-adenyl/thioesterforming enzymes, plays a critical yet unresolved role in SA metabolism (Nobuta et al., 2007; Warren et al., 1999). Interestingly, JAR1 (JASMONIC ACID RESPONSE LOCUS 1) of the same family, catalyzes formation of the hormone-amino acid conjugate JA-Ile, the active form of jasmonic acid (JA). JA-Ile binds to the E3 ubiquitin ligase F-box protein CORONATINE INSENSITIVE 1 (COI1) which targets a family of jasmonate ZIM domain (JAZ) transcriptional repressors for degradation via the 26S proteasome, thus resulting in activation of jasmonate responsive genes. (Chini et al., 2007; Thines et al., 2007; Staswick et al., 2002).  During SA-mediated immunity against (hemi)biotrophic pathogens, redox changes in the cytoplasm lead to monomerization of usually inactive complexed multimers of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) (Tada et al., 2008; Mou et al., 2003). Monomeric NPR1 relocates into the nucleus where it facilitates gene expression through interaction with TGA and possible recruitment of WRKY transcription factors (Wang et al., 2006; Zhang et al., 2003). TGA transcription factors bind to motifs in SA-inducible PATHOGENESIS-RELATED (PR) genes and other genes that are either dependent or independent of the regulator NPR1, consistent with the influence of multiple signalling routes over pathogen-induced genetic reprogramming (Fode et al., 2008; Thibaud-Nissen et al., 2006; Zhang et al., 1999). 12  Regulation of PR genes, which encode vacuole-targeted or secreted proteins with antimicrobial activities, is aided negatively by SNI1 and positively by RAD51, BRC2A and SSN2 (SUPPRESSOR OF NPR1, INDUCIBLE1; RAS ASSOCIATED WITH DIABETES 51D; BREAST CANCER 2A; SUPPRESSOR OF SNI1,2) (Song et al., 2011; Wang et al., 2010; Durrant et al., 2007; Mosher et al., 2006; Li et al., 1999; Van Loon and Van Strien, 1999). SNI1, a protein exhibiting structural similarity to Armadillo repeat proteins, functions as an inhibitor of gene expression. It is likely relieved through SA-induced activities of RAD51, SSN2 and TGA7 in the presence of NPR1, acting collectively as coactivators (Moore et al., 2011; Song et al., 2011; Pape et al., 2010).  Some components of the SA pathway are prone to modification by AtSIZ1 (A. THALIANA SAP and MIZ), the small ubiquitin-like modifier (SUMO) E3 ligase. Loss-of-function mutants of SIZ1 exhibit high SA levels and increased resistance to pathogens pointing towards a negative role for sumoylation in this branch of immunity (Lee et al., 2007). Post-translational addition of SUMO to proteins is reversible and may change abilities of modified proteins in molecular interactions (van den Burg et al., 2010).  NPR1-dependent expression of PR genes is essential for establishing systemic acquired resistance (SAR), a state of broad range immunity towards pathogens effective in both local and distal tissues. SAR is induced after a local infection and results in local and systemic resistance towards a broad range of pathogens. Recent evidence suggests that DIR1 (DEFECTIVE IN INDUCED RESISTANCE 1), in complex with a derivative of glycerol-3-phosphate, is the longdistance defence signal responsible for SAR outside of the local infection realm (Chanda et al., 2011; Maldonado et al., 2002). Levels of NPR1 in the nucleus are controlled through a CULLEN3-based E3 ligase complex which tags the protein for degradation. Dynamic amounts of NPR1 are important for proper induction of genes as well as inhibition of this activity in the absence of pathogens (Spoel et al., 2009). 13  1.6  Modifier of snc1 genes and the MOS4-associated complex  Genetic screens are a widely used approach to identify components in pathways of interest. In a standard screen, wild-type plants are mutagenized and desired mutants identified using selection criteria. Suppressor screens are a subcategory, in which the plants to be mutated are already mutants that exhibit phenotypes. Desired double mutants display suppression of mutant phenotypes and restored wild-type traits. Suppressor genes often function in the same pathway than the original mutated gene and can be identified using map-based cloning approaches (Zhang et al., 2007).  Functional involvement of a group of genes in plant immunity was discovered through a genetic suppressor screen based on the gain-of-function mutant suppressor of npr1, constitutive 1 (snc1) (Li et al., 2001). This mutant produces an altered form of a predicted TIR-NB-LRR immunity receptor which constitutively activates otherwise tightly regulated defence mechanisms. Redistribution of resources is likely responsible for the typical dwarf phenotype displayed by snc1 plants. However, the mutant is fertile and produces many siliques. snc1 also does not exhibit localized cell death which is a typical defence phenotype induced after detection of pathogen invasion and aimed at containing pathogen spread (Zhang et al., 2003; Li et al., 2001). Several types of mutagens were used to create second site mutations in the snc1 and snc1 npr1 mutant background, producing a collection of suppressor double mutants with largely restored wild-type phenotypes caused by mutations in modifier of snc1 (mos)genes (Monaghan et al., 2010).  Several MOS genes encode components of nucleo-cytoplasmic transport which was established as a major mechanism in defence responses. MOS3 and MOS7 share homology with nucleoporins, the main building blocks of the nuclear pore complex and which are required for mRNA export and for protein retention/export from the nucleus, respectively (Cheng et al., 14  2009; Zhang and Li, 2005). MOS6/IMPORTIN ALPHA 3 is an import receptor binding to the nuclear localisation signal of a nucleus-bound protein and aiding in crossing the nuclear envelope (Palma et al., 2005). MOS11 also contributes to mRNA export, potentially in the same pathway although upstream of MOS3 (Germain et al., 2010; Cheng et al., 2009; Zhang and Li, 2005). The importance of RNA pathways in defence is further supported through cloning of MOS2, which encodes a protein containing a G-patch and two KOW repeats, domains linked to m-RNA binding properties (Woloshen et al., 2011; Zhang et al., 2005).  A subset of snc1 suppressors are proteins aiding in post-translational modification processes such as farnesylation, which allows anchoring of a protein to the nuclear envelope. The βsubunit of farnesyltransferase, an enzyme that attaches the hydrophobic farnesyl group to proteins, is encoded by ERA1/MOS8 (Goritschnig et al., 2008). Plant defence also includes ubiquitination mechanism. MOS5 is allelic to UBA1, one of only two E1 ubiquitin activating enzymes in Arabidopsis. E1 enzymes initiate an ubiquitin conjugation cascade which often leads to a tagged proteins’ degradation via the 26S proteasome (Goritschnig et al., 2007). Participating in this pathway are also E3 ligases which are responsible for identification of proteins that should be ubiquitinated. The F-box protein CPR1 (CONSTITUTIVE EXPRESSER OF PR GENES 1) interacts with SNC1 in vivo and a related SKP1-CULLIN1-F-box (SCF) E3 ligase likely regulates relative levels of this and other R proteins via the ubiquitin proteasome pathway (Cheng et al., 2011; Hua and Vierstra, 2010)  The exact function of MOS4 is still unclear; however, it plays an important role as a core protein in the MOS4-associated complex (MAC). MOS4 co-purifies with 24 other proteins, most of which have homologs in human and yeast and are organized in similar aggregates (Johnson et al., 2011). Other MAC core proteins are AtCDC5/MAC1 (CELL DIVISION CYCLE 5), an R2-R3 Myb transcription factor, PRL1 (PLEIOTROPIC REGULATORY LOCUS 1), a WD40 protein likely providing structural support, and the redundant U-box (PUB) proteins MAC3A/MAC3B, 15  belonging to a class of single polypeptide E3 ligases (Monaghan et al., 2009; Yee and Goring, 2009; Palma et al., 2007). The human and yeast analogues of the MAC are associated with the spliceosome in their respective systems, a similar role is conceivable for the MAC. For example, the unequally redundant MAC5A/MAC5B proteins are putative RNA binding proteins (Monaghan et al., 2010). Importantly, MAC5B suppresses snc1 signalling and all MAC core proteins also display increased susceptibility towards a range of pathogens in single mutant analysis (Monaghan et al., 2010; Monaghan et al., 2009; Palma et al., 2007).  The snc1-induced defence array is built up through pathways utilizing key regulatory molecules NPR1 and SA, but signals are seemingly also routed through additional branches (Li et al., 2001). Double mutants of MOS4 and NPR1 exhibit increased vulnerability to pathogens compared to either of the single mutants, suggesting two independent affected pathways. Furthermore, expression of PATHOGENESIS RELATED 2, a hallmark of NPR1 regulation, is abolished in the snc1 mos4 mutant, further supporting MOS4 functionality in NPR1-independent signalling. Since SA levels are unaffected in atcdc5, mos4 and prl1 mutants, MAC signalling might be also routed through pathways independent to that of SA (Palma et al., 2007).  Characterization of several MAC components has revealed a complex picture of potential MAC functions; however immediate targets of the complex remain unknown  1.7  Pleiotropic regulatory locus 1 (PRL1)  PRL1 is one of the core components of the MAC and homologous to PRLG1, Prp46p and Prp5p/Cwf1p in H. sapiens, S cerevisae and S. pombe, respectively (Johnson et al., 2011). Large parts of the PRL1 amino acid sequence are arranged in WD40 repeats and folding into a seven-blade β-propeller, first described in the Gβ subunit of heterotrimeric G proteins (Xu and  16  Min, 2011). Through this interface, WD40 proteins participate in protein-protein, protein-peptide and protein-DNA interaction (Stirnimann et al., 2010).  Our current hypothesis of MAC organization assigns an important structural role to the PRL1 protein. Plants with mutations in PRL1 are severely susceptible to pathogens in addition to other pleiotropic effects (Palma et al., 2007). First identified as a mutant exhibiting growth arrest in media containing 6% sucrose, regulatory influence of PRL1 over a growing number of sugar, light, hormone, isoprenoid and stress-responsive pathways has been revealed (Flores-Perez et al., 2010; Baruah et al., 2009; Li et al., 2007; Abraham et al., 2003; Nemeth et al., 1998; Salchert et al., 1998). Morphologically, prl1 mutants exhibit phenotypes such as compromised root development, altered leaf morphology with serrated leaf margins, and an overall darker green colour, due to accumulation of isoprenoids such as chlorophyll (Flores-Perez et al., 2010; Nemeth et al., 1998).  PRL1 has been shown to directly bind AKIN10 and AKIN11 (ARABIDOPSIS SNF1 KINASE HOMOLOG 10/11) which are conserved protein kinases central to stress-, sugar- and developmental signalling (Baena-Gonzalez et al., 2007; Bhalerao et al., 1999). PRL1 inhibits AKIN kinase activities in vitro, potentially through a ubiquitination-based mechanism (Farras et al., 2001; Bhalerao et al., 1999). AKINs were co-purified with the α4/PAD1 (20S PROTEASOME ALPHA SUBUNIT PAD1) unit of the 26S proteasome, and both AKINs bind PRL1 with the same domain than used for interacting with the SKP1/ASK1 (ARABIDOPSIS SKP-LIKE 1) subunit of an SCF E3 ubiquitin ligase (Farras et al., 2001). A potential link between PRL1 and the plant ubiquitin machinery was further strengthened through co-precipitation of PRL1 with CULLEN4 (CUL4), a major scaffolding subunit of DCX-type E3 ligases, in vivo. Mutants of PRL1 and CUL4 exhibit similar phenotypes (Lee et al., 2008).  17  DCX-type E3 ligases are composed of several subunits. The CUL4 backbone interacts with the RING-finger protein RBX1 which in turn can bind an E2 protein; CUL4 also binds DDB1 (DAMAGED DNA BINDING 1) which recruits substrate receptors, bound to substrate, for ubiquitination (Biedermann and Hellmann, 2011; Vierstra, 2009). DDB1 is capable of interacting with a variety of substrate receptors through the 16aa DWD (DDB1 BINDING WD40) motif which is found in 78 proteins of rice and in 85 Arabidopsis proteins, including PRL1 (Lee et al., 2008). PRL1 features two DWD repeats imbedded into WD40 sequences which are needed to bind DDB1 in vivo, suggesting that PRL1 may act as a substrate receptor for DCX-type ubiquitination (Lee et al., 2008)  1.8  Research objectives  Much progress has been made in understanding the multitude of resistance mechanisms displayed by plants. Through progressive dissection of signalling pathways, it has also become clear that defence mechanisms are complex and new questions arise just after others have been answered. The MOS4-associated complex plays a role in effector-triggered immunity as well as in PAMP-triggered defences and, although advances in the characterization of MAC members have been made, the targets of the complex and its components remain elusive. A prominent member of the MAC is the WD40 protein PRL1. Comprised largely comprised of domains associated with protein-protein interactions, PRL1 is a good candidate for interactions with other defence molecules. PRL1 function is important to plant defence since mutant prl1 plants display severe susceptibility towards pathogens.  The research presented in this thesis describes a genetic suppressor screen aimed at identifying genes that function in PRL1-dependent resistance signalling. It also describes the cloning and subsequent characterization of two such genes, PRL2 and SOP2. Based on  18  previous experience with screens carried out in our laboratory, the following objectives were established:  1) Compilation and preliminary analysis of suppressor of prl1 (sop) mutants 2) Analysis of the relationships between PRL1 and homologous gene, PRL2 3) Characterization of SOP2, a member of a novel gene family with unknown function  To address the objectives, a combination of approaches including screening methodologies, positional cloning, infection assays and PCR-based protocols were used.  1.8.1 Objective 1: Compilation and preliminary analysis of suppressor of prl1 (sop) mutants We previously identified a number of defence signalling components by means of a similar suppressor screen based on the plant resistance gene snc1 (Monaghan et al., 2010). The research described in Chapter 2, The prl1 suppressor screen results in twenty-two mutants with defects in candidate genes, aimed to identify prl1 suppressor mutants. These suppressors were expected to no longer exhibit prl1-associated traits but morphologically resemble a wild-type plant. Through backcrosses with the original prl1 mutant, the recessive, dominant or semidominant nature of the respective suppressing mutation was evaluated. To test whether the mutations affect defence, obtained morphological suppressors were subsequently subjected to a second screening step in which defence responses towards oomycete pathogen Hyaloperonospora arabidopsidis EMWA1 were tested.  19  1.8.2 Objective 2: Analysis of the relationships between PRL1 and homologous gene, PRL2 Using map-based cloning methods and sequencing, we identified a regulatory gain-of-function allele of PLEIOTROPIC REGULATORY LOCUS 2 (PRL2) as dominant suppressor of prl1 phenotypes. Based on high sequence similarity, this gene had been previously documented as a homolog of PRL1, however, due to very low expression levels, has not been studied yet (Nemeth et al., 1998). Due to the regulatory nature of the discovered mutation in the prl2-1D mutant, we were in a unique position to analyse the relationship between the two related proteins and test whether they carry out similar functions. In a comprehensive approach, we examined defence abilities directed against both virulent and avirulent pathogens as well as developmental characteristics such as root length and flowering time. This work is described in Chapter 3, A gain-of-function mutation in PLEIOTROPIC REGULATORY LOCUS 2 reveals unequal redundancy and functional equivalence between the WD40 protein and close homolog PRL1.  1.8.3 Objective 3: Characterization of SOP2, member of a novel gene family with unknown function sop2-1D is the second dominant mutant derived from our screen, exhibiting fully restored wildtype appearance and resistance. To identify the responsible mutation, we followed a positional cloning approach with subsequent sequencing of candidate genes. Intriguingly, we determined that an unknown gene functions in PRL1 signalling. This finding led us to ask a number of questions: What causes the dominant nature of the mutation? Do homologs of SOP2 exist in Arabidopsis, in other plants, in other kingdoms? What is the subcellular localization of the encoded protein? Is SOP2 required for plant resistance and what can we learn about its function? And also, is SOP2 a target of the MAC? In Chapter 4, A dominant mutation in an 20  uncharacterized gene identifies a component of PRL1 signalling specific to the plant kingdom, the cloning and characterization of SOP2 is described. In this process, we used methods including PCR-based techniques, infection assays with pathogens and a number of publicly available computer algorithms.  21  fungal/oomycete pathogen apoplast  R  cytoplasm  R R protein stabilizing complex  modification of host proteins  R protein activation recognition of guarded host proteins d e re igg P-tr g PAM ignalin s  repressed R protein  some defense components  direct recognition of effectors  effector-triggered signaling some R-proteins  negative regulator  nucleus  transcription factors PAMP-triggered immunity  effector-triggered immunity  plasma membrane  defense gene expression  RESISTANCE  Figure 1. Signalling events involved in plant innate immunity Plants have evolved the ability to perceive highly conserved pathogen-associated molecular patterns (PAMPs) via transmembrane pattern recognition receptors (PRRs). PRR activation triggers mitogenactivated protein kinase (MAPK) signaling cascades that induce defense gene expression and hinder the growth of some microbial populations. During infection, pathogenic microbes deliver effector proteins into host cells, where they function to suppress or interfere with PAMP-triggered immunity and other defense responses. In resistant plants, cytoplasmic and membrane-associated resistance (R) proteins recognize effectors either directly or indirectly through the surveillance of guarded plant proteins and trigger effector-triggered immunity. Activated R proteins result in genetic reprogramming and pronounced physiological changes in the infected plant cell that ultimately result in resistance. Adapted from Monaghan et al. 2009.  22  2  A prl1 suppressor screen yielded twenty-two  mutants with defects in candidate genes 2.1  Introduction  Plant resistance is conferred by preformed barriers and components, broad range detection of microbes and pathogen-specific defence mechanisms (Jones and Dangl, 2006; Heath, 2000; Osbourn, 1996). If pathogens overcome these layers of protection, ensuing disease symptoms will affect crop yield and horticultural success. To reduce or prevent economic losses, a comprehensive understanding of natural plant defence mechanisms is needed. However, efforts are impeded by the complex genomes of important cultivars such as broccoli, rape seed and cauliflower, (members of the Brassicaceae family). Research focussing on the related, but more simply organized model plant Arabidopsis thaliana has significantly advanced our knowledge of plant resistance mechanisms, not least due to the feasibility of mutant screens in this plant (Malinovsky et al., 2010; Zhang and Li, 2005; Glazebrook et al., 1996; Glazebrook and Ausubel, 1994).  In forward genetic screens, radiation or chemicals such as ethyl methanesulfonate (EMS) are often used as mutagens and resulting mutants with defects in defence responses can be identified. In Arabidopsis and other plants, insertional mutagenesis methods mediated by Agrobacterium tumefaciens are also an option. In a suppressor screen, mutants are mutagenized a second time and screened for desired double mutants exhibiting reverted wildtype attributes, often due to an additional mutation in the same signalling pathway. In our laboratory, a number of previously unknown resistance components have been identified as suppressors of snc1, a mutant allele of a predicted resistance gene (Germain et al., 2010; Monaghan et al., 2010; Zhang et al., 2003). An in depth analysis of MOS4 (MODIFIER OR 23  SNC1,4) revealed the existence of a MOS4-associated complex (MAC), playing a role in plant defence (Johnson et al., 2011; Monaghan et al., 2009; Palma et al., 2007) The MAC displays attributes of a nodal point, channeling signals initiated by both major types of R proteins and routing through pathways both dependent and independent of the marker molecules salicylic acid (SA) and NPR1 (NONEXPRESSOR OF PATHOGENESIS RELATED PROTEINS1) (Palma et al., 2007). Signalling steps controlled by the MAC will be best understood through the identification of targets, which remain elusive to this point.  Counting as one of the five core members of the MAC, the WD40 repeat protein PRL1 (PLEIOTROPIC REGULATORY LOCUS 1) belongs to a family with prominent protein-protein interactions domains (Stirnimann et al., 2010; Nemeth et al., 1998). Importantly, mutations in PRL1 result in substantially compromised plant resistance (Palma et al., 2007). Both characteristics make PRL1 a promising candidate for a suppressor screen with the goal of finding downstream components of MAC-related defence signalling. Using EMS as a mutagen, we induced random second site point mutations in T-DNA mutants of prl1 (prl1-2) and screened progeny for morphological as well as defence-related phenotypes. The goal was to identify novel elements in PRL1 mediated signal relay by cloning of obtained suppressors of prl1 (sop) mutations.  2.2  Material and methods  2.2.1 Plant material and growth Wild-type Arabidopsis ecotypes Columbia (Col-0), Landsberg erecta (Ler) and derived mutants were usually grown on soil in a 16h light / 8h dark regime. The T-DNA mutants prl1-2 (Salk_008466) and prl1-3 (Salk_039427) were obtained from the Arabidopsis Biological Research Centre and genotyped by PCR using insertion flanking oligonucleotides PRL1-Salk24  NF (5'-GATGAAAGTTGCGTTTGGAG-'3) and PRL1-NR-A (5’-ACTACCTACACTACCTAGAGC‘3).  2.2.2 Agrobacterium tumefaciens mediated mutagenesis Agrobacterium tumefaciens mediated T-DNA mutagenesis was carried out on approximately 128,000 prl1-2 and prl1-3 mutant plants. M1 and M2 progeny from transformed plants was treated with the herbicide BASTA ® to select for successful genomic integration of the T-DNA, encoding the relevant resistance gene.  2.2.3 EMS mutagenesis and primary morphological screen Approximately 25,000 prl1-2 seeds (0.5g) were subject to EMS treatment (The Arabidopsis Information Resource (TAIR), [http://www.arabidopsis.org/comguide/chap_1_plants/6_EMS_mutagenesis.html]). Resulting M1 plants were allowed to self and M2 seeds harvested into 79 pools each containing seeds of 2025 plants. Approximately 500 seeds per M2 pool were either screened on soil or alternatively on MS plates containing 6% sucrose. prl1 mutant plants germinate late and display characteristic serrated and dark green leaf morphology.Tthese symptoms intensify in high sugar conditions finally resulting in growth arrest (Nemeth et al., 1998) .  During the primary screen based on suppression of described phenotypes, between one and eleven candidates from 29 M2 seed pools, totalling 86 candidate plants, were transplanted. Using the primers PRL1-Salk-NF and PRL1-NR-A, homozygous prl1-2 background was confirmed and contaminants discarded. Morphological verification of M3 and M4 plants excluded some lines with low levels of suppression. Forty-nine putative mutants from 22 pools were obtained and a representative for each pool chosen. The selected mutants were named 25  according to their respective pools P1 through P71, followed by the nomination “A” for plate grown, “B” for soil grown and a number.  2.2.4 Secondary resistance screen Hyaloperonospora arabidopsidis EMWA1 (formerly Peronospora parasitica and Hyaloperonospora parasitica) is a biotrophic oomycete pathogen which causes downy mildew on Arabidopsis (Holub, 2008; Slusarenko and Schlaich, 2003). It is an obligate pathogen which is propagated on the susceptible Ws ecotype usually completing a life cycle in one week at 16ºC and high humidity (Li et al., 2001). For the secondary screen based on disease resistance traits, two-week old soil-grown progeny of confirmed sop mutants were infected with a conidiospore solution of H.a. EMWA1 (1 x 106 spores/ml) and disease ratings (n=3) were assessed 7 days post-infection (DPI). Using a five category ranking system, resistant Col-0 is scored as “1”, highly susceptible Ws as “5” and prl1-2 as “4”. Suppressor mutants were classified as resistant with a ranking of “1-2” and classified as susceptible when scored “3” or higher.  2.2.5 Assessment of genetic inheritance To assess the genetic nature of sop mutations, backcrosses between the sop prl1-2 and homozygous prl1-2 single mutants were carried. In such a backcross, the prl1-2 mutation is fixed and only the suppressing locus is segregating. Homozygous recessive sop mutations resulted in exclusive prl1 like progeny, whereas homozygous dominant sops gave only wild-type like backcross progeny. Phenotypically intermediate progeny indicated semi-dominant (incomplete dominant) inheritance of the respective sop mutation.  26  2.3  Results  2.3.1 Complete and partial suppression of prl1 phenotypes by sop mutations The recessive T-DNA mutant prl1-2 displays a number of visible phenotypes including serrated leaf margins, short roots and growth arrest under high sugar conditions (Nemeth et al., 1998). We screened for suppressor or prl1 (sop) mutants with restored wild-type morphology among M1 and M2 progeny of the T-DNA screen and in the M2 generation of the EMS screen. We identified 22 sops from EMS M2 pools and confirmed suppression through examination of progeny in following generations whereas our T-DNA approach was not successful. Among the mutants that were obtained, five showed complete suppression of the assayed prl1-associated phenotypes (prl1-2 sop1, prl1-2 sop2, prl1-2 sop8, prl1-2 sop13 and prl1-2 sop15); seventeen additional mutants exhibited partial suppression (Tab. 1 and Fig. 2).  2.3.2 Restored R protein mediated resistance in most prl1-2 sop mutants Resistance protein mediated signalling is impaired in single mutants of prl1-2, resulting in successful growth of the avirulent oomycete Hyaloperonospora arabidopsidis (H.a.) EMWA1 compared to resistant Col-0 wild-type plants (Palma et al., 2007). When prl1-2 sop suppressor mutants were challenged with a high dose (150,000 spores/ml) of H.a. EMWA1, 19 mutants displayed restored wild-type resistance. The remaining three mutants (prl1-2 sop9, prl1-2 sop16 and prl1-2 sop17) allowed pathogen colonization at a level that was similar to prl1-2 single mutants (Tab. 1).  27  2.3.3 Suppressor phenotypes are caused by dominant, semi-dominant and recessive mutations Backcrossing prl1-2 sop mutants to the prl1-2 parent according to standard Mendelian tests allowed us to determine the inheritance pattern of the sop alleles. Exclusively prl1-like progeny in the F1 generation of such a cross would provide evidence for a recessive sop allele that is present for both copies of the gene (homozygous). Recessive patterns were obtained for nine suppressors (Table 1). In contrast, progeny consisting of nine prl1- like and twelve wild-type plants were obtained after back-crossing a phenotypically wild-type prl1-2 sop2 individual. This ratio suggests dominant inheritance of sop2 and heterozygous gene configuration at the sop2 locus in the tested specimen (expected 1:1, χ2 = 0.42, P = 0.51). Dominant inheritance was also established for sop1 since only wild-type progeny (26 plants) resulted from the backcross. sop1 and sop2 are different genetic loci and located on chromosome three and two, respectively, as was determined in preliminary mapping analysis. For suppressors sop15, sop 17 and sop10, a semi-dominant (incomplete dominant) relationship is most likely because the backcross progeny exhibited phenotypes intermediate to both parents (Table 1). Several sop lines were investigated further in our collaborating laboratory at the National Institute for Biological Sciences (NIBS, P.R.China), where approximate numbers of prl1-like progeny in the F2 of mapping crosses were determined. The observed F2 segregation ratios support inheritance patterns obtained through backcrossing in most cases; i.e., for recessive mutants approximately 1/16 of F2 progeny should be prl1-like. Contradicting results were obtained for sop11 and sop20, which have been listed as non-classified mutants (Tab. 1).  2.4  Discussion  Using EMS as mutagen, we succeeded in producing more than twenty prl1 suppressors harbouring mutations in candidate genes relevant to the PRL1 signalling branch. Analysis of 28  prl1-typical attributes such as pointy, serrated leaves and dark green colour allowed us to efficiently process M2 pools since morphological suppressors were clearly visible due to fully or partially restored wild-type phenotypes. The isolated mutants were designated suppressor of prl1 (sop).  As previously noted, mutations in PRL1 impact transformation efficiencies (Nemeth et al., 1998). Mutagenesis mediated by A. tumefaciens is based on a transformation protocol and thus our lack of success in producing suppressors with insertional mutations can at least be partially explained through low transformation efficiency. Odds of a successful insertion were likely further reduced through additional biotic stress caused by greenhouse pathogens. The chemical agent EMS, which is used on Arabidopsis seeds thus represents the better choice in mutagen when dealing with stress and disease-sensitive mutants. Observations made during the T-DNA screen resulted in a modified transformation protocol, which was successfully employed for cloning of sop2 (see Chapter 4 and Appendix 1).  After exposure to an established Arabidopsis pathogen, most sop mutants displayed restored R protein mediated resistance, independent of the level of morphological suppression. Whereas limits in quantification of resistance cannot be excluded, these results might suggest that PRL1related morphology and resistance are not fully linked. In a reverse example, sop17, which is highly susceptible to pathogen H.a. EMWA1, shows largely restored wild-type morphology. Next to an important role in plant defence, PRL1 has been reported to function as a regulator of sugar, hormone, light and O2 responsive genes (Flores-Perez et al., 2010; Baruah et al., 2009; Bhalerao et al., 1999; Nemeth et al., 1998; Salchert et al., 1998). It is tempting to hypothesize that sop17 and partial suppressors such as sop10 or sop12 might signal in parallel or through shared components relative to defence signalling.  29  The prl1 suppressor screen resulted in 13 recessive mutants, more than 70% of all classified suppressors. Screens usually result in a majority of recessive mutants since most genes in Arabidopsis are haplosufficient, able to sustain wild-type function even if one allele is defective. Since EMS induces point mutations, functionality of a protein can be affected on a quantitative scale which manifests as a partial or fully suppressed phenotype in a homozygous suppressor mutant plant. If a member of a partially redundant gene family is affected, the effect could be similar since the remaining family genes still provide most of the functionality needed. A different type of dosage effect is often responsible for semi-dominant mutations, which make up 16% of obtained mutants. A portion of Arabidopsis genes are haplo-insufficient, referring to the need for product produced by both gene alleles to confer normal function. If impairment of one allele reduces product levels below a critical threshold, a mutant phenotype is visible which is intensified if both copies are affected. In essence, these are dominant loss-of-function mutations.  The smallest number of sop mutants fall into the dominant class. Working with non-recessive mutants is challenging since genetic analysis, complementation tests and pathway classification through epistasis are more time consuming and often difficult. However, the study of dominant mutations can lead to unique insights into biological proceedings and both sop1 and sop2 have been studied in detail in Chapters 3 and 4.  All suppressors were isolated from separate screening pools and since both dominant mutations have already been mapped to different areas of the genome, other independent and potentially novel genetic loci may be found among the yet uncharacterized prl1 suppressors. Similar looking sop mutants such as sop5 and sop19 or sop4 and sop10 might harbour mutations in the same respective gene, however, comprehensive complementation tests are required to determine the actual number of affected loci among the suppressor mutants.  30  Table 1. Analysis of sop mutants  Screening namea  22 sops  Morphological suppressionb  Backrossc (N⁰ of plants)  N⁰ in F2 of mapping cross (prl1like)d  H.a. EMWA1 resistante  Chr.  n.d. n.d.  Yes Yes  3 2  1/16 n.d. n.d.  Yes No Yes  <10/400 ~20/500 ~40/400 <10/400 ~10/200 n.d. n.d. n.d. ~10/300 ~40/400 ~40/400 n.d. ~10/400  Yes Yes Yes Yes Yes No Yes Yes Yes Yes No Yes Yes  n.d. 25% n.d. ~10/400  Yes Yes Yes Yes  Dominant mutants obtained P2-A2 P20-A4  sop1 sop2  complete complete  WT (26) prl1 (9), WT (12)  Semi-dominant mutants obtained P33-A1 P38-A1 P48-A1  sop15 sop17 sop19  complete complete partial  neither (3) neither (49) neither (10)  Recessive mutants obtained P6-A1 P12-B2 P13-B2 P16-A1 P18-A7 P21-A1 P23-A1 P27-B1 P28-B1 P31-B2 P34-B1 P67-A1 P71-A1  sop3 sop5 sop6 sop7 sop8 sop9 sop10 sop12 sop13 sop14 sop16 sop21 sop22  partial partial partial partial complete partial partial partial partial partial partial partial partial  neither (24) n.d. n.d. prl1 (26) n.d. prl1 (21) prl1 (2) prl1 (32) n.d. prl1 (17) prl1 (45) prl1 (6) prl1 (6)  Non-classified mutants P7-B1 P25-A1 P43-A1 P61-A1  sop4 sop11 sop18 sop20  partial partial partial partial  prl1-2 WT  n.d. prl1(26) n.d. neither (27)  No Yes  a  Identification name in original screen Morphological suppression levels varied significantly. A mutant was declared a complete suppressor if WT-like colour, root length and leaf morphology was exhibited. c Number of progeny and their phenotypes obtained through backcrossing of respective prl1-2 sop mutants with prl1-2 single mutant d Approximate number of prl1-like progeny obtained in F2 of mapping population e Suppression of prl1- related disease susceptibility was quantified using avirulent pathogen H.a. EMWA1, see Materials and Methods b  WT, wild-type  n.d., not determined  31  sop1  sop2  sop13  sop14  sop3  sop4  sop15  sop16  sop5  sop6  sop17  sop18  sop7  sop8  sop19  sop20  sop9  sop10  sop21  sop22  sop11  sop12  prl1-2  Col (WT)  Table 2. sop mutants exhibit complete and partial suppression of prl1 phenotypes Most images were taken when plants were four weeks old. Images of sop2, sop3, sop13, sop21, sop22, Col-0 and prl1-2 show five-week-old plants.  32  3  A gain-of-function mutation in PLEIOTROPIC  REGULATORY LOCUS 2 reveals unequal redundancy and functional equivalence between the WD40 protein and the close homolog PRL1 3.1  Introduction  To colonize a plant host, pathogens need to overcome an array of defence mechanisms (Jones and Dangl, 2006). The presence of microbes at the plant surface is detected by pattern recognition receptors that recognize pathogen-associated molecular patterns (PAMP) and a general resistance reponse is induced. Pathogenic microbes are able to evade or suppress PAMP-triggered immunity (PTI) by delivering effector molecules into the host cell which interfere with PTI signalling. Activity of such pathogen-derived effector molecules is detected by the second layer of plant defence, a surveillance system comprised of resistance (R) protein complexes which, upon induction, signal to establish local and systemic resistance (ETI) (van der Hoorn and Kamoun, 2008).  Most R-proteins use NDR1 (NONRACE-SPECIFIC DISEASE RESISTANCE) (Knepper et al., 2011; Century et al., 1995) or the EDS1/PAD4 node (ENHANCED DISEASE SUSCEPTIBILITY1/PHYTOALEXIN DEFICIENT4) (Wiermer et al., 2005; Feys et al., 2001) early in signalling and before the onset of salicylic acid (SA) production which is mediated by SID2 and EDS5 (SA INDUCTION DEFICIENT2, ENHANCED DISEASE SUSCEPTIBILITY5)(Nawrath et al., 2002; Wildermuth et al., 2001). One pathway downstream of SA accumulation requires the regulatory protein NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1) to induce defence gene expression through interaction with TGA transcription factors (Zhang et al., 2003; Cao et al., 1994). At least two other 33  pathways are used by R proteins which either do not require NPR1 (Bowling et al., 1997) or bypass both SA production and the later involvement of NPR1 (Shah et al., 2001). Virulent pathogens evade specific detection and can colonize the plant, yet their presence is still perceived causing a weaker and delayed (basal) response overlapping in parts with R protein mediated signalling (Qi et al., 2011; Zhang and Li, 2005; Navarro et al., 2004)  Substantial involvement of the nucleo-cytoplasmic transport machinery in plant resistance was unveiled through characterization of mos (modifier of snc1) mutants which had been obtained through a suppressor screen based on snc1, a constitutively signalling predicted resistance gene, (Zhang et al., 2003). MOS3 and MOS7 are nuclear pore components, MOS6 and MOS11 are facilitators of protein import and mRNA export respectively (reviewed in (Germain et al., 2010; Monaghan et al., 2010)). Comprehensive analysis of the recessive mos4 mutation revealed an important role in plant signalling for the evolutionary conserved MOS4-associated complex (MAC) which consists of approximately 20 proteins (Johnson et al., 2011; Monaghan et al., 2009; Palma et al., 2007). Homologous complexes in yeast and humans function in premRNA splicing (Ajuh et al., 2000; Cheng et al., 1993; Tarn et al., 1993).  The MAC functions in signal relay mediated by both major classes of resistanc proteins. Interestingly, the less understood SA - and NPR1 independent signal routes are also used in the relay of defence information downstream of the complex. In single mutant analysis, all three firstly described MAC components display impaired PTI and ETI however defects in PLEIOTROPIC REGULATORY LOCUS 1 (PRL1/MAC2) reduce plant resistance levels subtantially more than do mutations in MOS4 or CELL DIVISION CYCLE 5 (AtCDC5/MAC1) (Palma et al., 2007; Nemeth et al., 1998). PRL1 has been implicated in the modulation of sugarmediated, hormone and stress regulated responses (Nemeth et al., 1998; Salchert et al., 1998). In the MAC, this WD40 protein is hypthesized to provide structural support consistant with abilities of this domain family to facilitate transient or stable interactions between proteins 34  (Stirnimann et al., 2010). To better understand PRL1 function in plant biology, we conducted a suppressor screen in the prl1 mutant background. Characterization of the dominant sop1 (suppressor of prl1, 1) mutant revealed functional redundancy between the versatile PRL1 and its homolog PRL2.  3.2  Materials and methods  3.2.1 Plant material and growth Wild-type Arabidopsis ecotypes Columbia (Col-0), Landsberg erecta (Ler) , Wassilewskija (Ws) and derived mutants were grown on soil in a 16h light / 8h dark regime. T-DNA mutants prl1-2 (Salk_008466), prl2-2 (Salk_075970), prl2-3 (Salk_133878) were obtained from the Arabidopsis Biological Research Centre and genotyped by PCR using insertion flanking oligonucleotides PRL1-Salk-NF (5'-GATGAAAGTTGCGTTTGGAG-'3) and PRL1-NR-A (5’ACTACCTACACTACCTAGAGC-‘3) for prl1-2 and PRL2-Salk-F (5'TCTGAACCGACGCTTAATGAG-'3) and PRL2-Salk-R (5'-TGTAGGCTTACTTGCAGGTTC-'3) for prl2-2 and prl2-3.  3.2.2 Morphological characterization For root assays, seeds were vernalized for three days at 4ºC and grown for seven days on Murashige and Skoog (MS) medium. Square plates were kept upright in a growth rack and exposed to 16h light and 8h dark per day. Root lengths of 10 individual plants per genotype were scored using a small ruler. To evaluate flowering time, seeds were vernalized for at least three days and plants grown under long day conditions (16h dark/ 8h light). Plants were observed daily and onset of flowering was established when an emerging flower measured 1cm in height. Rosette leaves present at the time of bolting were counted for 10 plants per genotype. 35  3.2.3 Infection assays Pseudomonas syringae pathovar maculicola (P.s.m.) ES4326 and pathovar tomato (P.s.t.) DC3000 are hemi-biotrophic bacterial pathogens responsible for leaf spot and bacterial speck disease on Arabidopsis, respectively. The bacteria were grown at 28-30ºC in liquid LB or LB plates containing 50 mg/ml Streptomycin (P.s.m. ES4326) or 25 mg/ml Rifampicin and 50 mg/ml Kanamycin (P.s.t. avrPphB and avrRps4). Arabidopsis plants are inoculated using a small needless plastic syringe with the opening lightly pressed against an Arabidopsis leave underside allowing injection of bacteria into the plant apoplast. A low dose (OD 600= 0.0001), referred to as enhanced disease susceptibility (EDS) dose, was used in virulent (P.s.m. 4326) infections. A higher dose (OD600= 0.002) was employed with avirulent P.s.t. avrPphB and avrRps4. On the first day and three days after inoculation, leaf discs of 0.32cm 2 were cut with a standard paper hole-punch, samples were homogenized in 10 mM MgCl 2 and a series of six dilutions was plated. Bacterial colony forming units (cfu) were calculated after two days of incubation at 28ºC.  For H.a. EMWA1 infection assays, 2.5 week old plants were spray-inoculated with a low dose (50,000 spores/ml) of conidiospores to determine resistance defects in a compatible interaction, a high dose (100,000 spores/ml) was used to evaluate incompatible interactions. After 7-10 days, plants were harvested in a 50 ml Falcon tube containing up to 5 ml water, conidiospores were released using a vortex for 10 seconds and quantified using a hemocytometer.  3.2.4 Positional cloning Molecular markers for map-based cloning were PCR based and detected either length (InDel) or single nucleotide polymorphisms (SNP) between Col-0 and Ler ecotypes (Monsanto Arabidopsis Ler sequence available at TAIR: 36  [http://www.arabidopsis.org/browse/Cereon/index.jsp]). When employing InDel markers, one set of primers is used for both ecotypes and amplified fragments are visualized on 1-2% agarose gels. For SNP markers, two forward primers differing in the last two nucleotides were designed allowing ecotype specific binding as well as pairing with a common reverse primer (Bui and Liu, 2009). The two primer combinations are used in alternating reactions on genotypes and fragments are analysed on 1% agarose gels. For primer sequences, see Appendix 2.  3.2.5 Quantification of PRL2 mRNA levels Seeds of the genotypes prl1-2, prl1-2 prl2-1d, prl2-1d, prl2-2 and Col-0 wild-type were vernalized for 7 days and plated on 0.5 MS containing 100 mg/ml Ampicillin. The plates were incubated in a growth chamber for ten days using a 16h light, 8h dark regime. Tissue of 10-dayold seedlings was collected in a 2ml reaction tube containing two glass beads and frozen immediately. RNA was extracted using the Totally RNA kit (Ambion) and Reverse Transcriptase (SuperScript II, Invitrogen) was used to produce c-DNA copies of the transcriptome Relative amounts of PRL2 cDNA (PRL2-RT-F: 5’-CGTAATGGTCACTGGAGGTG-‘3, PRL2-RT-R: 5’TTTTTCTGGCTTCGAGTTTGA-‘3) and Tubulin (control) c-DNA (5′ACGTATCGATGTCTATTTCAACG-3′ and 5′-ATATCGTAGAGAGCCTCATTGTCC-3′) present in the collected tissues were quantified using real-time PCR.  3.2.6 Single and double mutant construction For creation of the sop1-1d/prl2-1d single mutant, homozygous prl1-2 sop1-1d mutants were crossed to Col-0 wild-type plants and the single was identified in the F2 generation using allele specific primers P2A2-M-F2 (5’-GTCGGATAAAATCCTATTTGT-3’) and P2A2-WT-R (5’GCGAAACTGTTGATTAACCT-3’). To obtain the prl1-2 prl2-2 double mutant, homozygous prl11 and prl2-2 plants were crossed and double mutants were confirmed in the F2 using insertion 37  flanking primers PRL1-Salk-NF and PRL1-NR-A for prl1-1 and PRL2-Salk-F and PRL2-Salk-R for prl2-2. The prl1-2 prl2-2 mutant was previously isolated by Dr. Palma in our laboratory.  3.2.7 Transformation of JAtY clones The genomic JAtY library was created at the John Innes Centre (UK) and is based on the pYLTAC17 vector allowing for the selection of transformed plants through BASTA selection. Populations of flowering prl1-2 and prl1-12 sop1-1d plants were subject to Agrobacterium tumefaciens-mediated transformation and seeds collected after six weeks. T1 progeny was sprayed 3-5 times with BASTA® in the first two weeks after germination and transformants identified after three weeks.  3.3  Results  3.3.1 sop1-1D fully suppresses prl1-associated phenotypes Morphological traits associated with the T-DNA mutant of PRL1 (prl1-2) such as serrated leaf margins, pointy shaped first leaves and darker leaf colour, are no longer exhibited by the prl1-2 sop1-1D suppressor mutant which instead resembles a wild-type plant (Fig. 2A). When comparing average root lengths of Col-0 (WT), prl1-2 and prl1-2 sop1-1D plants, double mutants grow roots of wild-type length instead of short roots typical for prl1 mutants (Fig. 2B) (Nemeth et al., 1998). sop1-1D also suppresses early flowering in the double mutant since prl12 sop1-1D mutants initiate flowering with twice as many rosette leaves present than prl1-2 single mutants (Fig. 2C). Dominant suppression of prl1 phenotypes by sop1-1D was demonstrated by backcrossing of a pure breeding prl1-2 sop1-1D line to the prl1-2 parent which resulted in 26 F1 plants with wild-type morphology (Tab.1).  38  For evaluation of defence responses in Arabidopsis plants, we employ both virulent and avirulent pathogens in our laboratory, with the former successfully colonizing the plant and raising only insufficient defence reactions whereas the latter is recognized by specialized immunity receptors and prevented from causing further harm. Using virulent pathogen Pseudomonas syringae pathovar maculicola (P.s.m) ES4326 and avirulent Hyaloperonospora arabidopsidis (H.a.) EMWA1, substantial differences both in basal defence and R protein mediated resistance between the prl1-2 sop1-1D double mutant and the original prl1-2 single mutant were observed. The characteristic enhanced disease susceptibility (EDS) in basal defence, usually displayed by prl1-2 plants when exposed to the virulent bacterial pathogen, cannot be detected in the double mutant (Fig. 2D) (Palma et al., 2007). Similarly, effective resistance signalling mediated by the R protein RESISTANCE TO PERONOSPORA PARASITICA 4 (RPP4) ensures resistance towards oomycete H.a. EMWA1 in both Col-0 and prl1-2 sop1-1D plants, thereby overcoming the compromised response of the prl1 background mutation (Fig. 2E). Morphological assessments and pathogen infection assays thus identify sop1-1D as a full suppressor of prl1- related phenotypes.  3.3.2 Map-based cloning of sop1-1D identifies a molecular lesion in PLEIOTROPIC REGULATORY LOCUS 2 (PRL2) The sop1-1D prl1-2 double mutant (Col-0 background) was crossed with the Ler ecotype to create segregating progeny, a requirement for map-based cloning techniques. In the F2 generation of the mapping cross, analysis of 48 plants with prl1 mutant morphology identified the approximate location of sop1-1D between Insertion/Deletion (InDel) markers MIE1 (4.87Mb) and MRC8 (6.21Mb) on the top arm of chromosome 3 (Fig. 3A). Progeny of prl1-2 homozygous F2 plants with heterozygosity for the sop1-1D locus was used in further mapping steps. In total, 1008 F3 plants were examined and 61 recombinants for above markers of either prl1-2 or wildtype morphology identified. For recombinants, allele configuration at the sop1-1d locus was 39  confirmed using morphological segregation patterns in the following (F4) generation. Using the selected recombinants, the sop1-1D locus was further mapped to a region flanked by the InDel markers MDC8 (5.58Mb) and K14A17 (5.84Mb) and was finally included in a 62kb section on Chromosome 3 between InDel marker MGL6 and single nucleotide polymorphism (SNP) marker MGL6-SNP5, with two recombinants for each marker remaining. The genomic area between 5.63Mb (MGL6) and 5.69Mb (MGL6-SNP5) on the top arm of Chromosome 3 harbours 20 genes (Fig. 3A (Huala et al., 2001)), among them At3g16650 encoded PLEIOTROPIC REGULATORY LOCUS 2 (PRL2) which is homologous to described WD40 family protein PRL1 (Nemeth et al., 1998). Through sequencing of the At3g16650 open reading frame in the prl1-2 sop1-1D mutant, a C to T substitution was identified in the first exon of the PRL2 gene, 58bp upstream of the translational start codon (Fig. 3B).  During mapping of sop1-1D, we encountered marker patterns indicative of an area with low recombination frequency on chromosome I, co-seggregating with prl1 mutant phenotypes and correlating with Col-0 morphology. Initially, this led us to hypothesize an ecotype specific suppressor gene in Ler plants. Such specificity is not unknown; for example, the Arabidopsis ecotype Ws is insensitive to flagellin presence since it does not encode a functional FLS2 receptor (Gomez-Gomez et al., 1999). Comprehensive analysis of this region however led us to conclude the presence of a chromosome mutation. During the initial creation of prl1-2 using TDNA mutagenesis, a fragment of chromosome IV, encompassing the mutated PRL1 locus, appears to have been relocated to chromosome I. We did not determine the exact scope or location of the assumed translocation; however, molecular marker-based flanking suggests insertion between 4.15Mb and 4.99Mb on chromosome I (data not shown). A genomic rearrangement can cause improper pairing of chromosomes during meiosis, resulting in the loss of gametes and which would manifest in observed reduced numbers of recombinants. Interchromosomal rearrangements, deletions or insertions have been previously associated with  40  T-DNA mutagenesis and are an unfortunate shortcoming of this technique (Tax and Vernon, 2001).  3.3.3 sop1-1D is a gain-of-function allele of PRL2 PRL2 has been previously reported as the homolog of PRL1 in Arabidopsis (Baruah et al., 2009; Nemeth et al., 1998). Public data from the transcriptome platform AtGenExpress (http://jsp.weigelworld.org/expviz/expviz.jsp (Schmid et al., 2005) ) identifies on average 80% lower expression of the PRL2 gene compared to PRL1 across tested developmental stages in the database (Appendix 3). Similar expression patterns are also detected in response to abiotic stress, hormones and pathogens (Appendix 3). The encountered mutation in prl1-2 sop1-1D is located in the 5’ untranslated region (5’UTR) of PRL2, thus excluding changes to the protein structure. Consequently, we hypothesized a regulatory effect, potentially impacting PRL2 transcript levels. We examined seedling tissues of prl1-2 sop1-1D and sop1-1D mutants and detected PRL2 cDNA levels approximately twice as high in prl1-2 sop1-1D compared to the prl1-2 single mutant and wild-type controls as determined by quantitative reverse transcription (RT)-PCR analysis (Fig. 3C,D) and as validated by semi-quantitative PCR-analysis using primers PRL2-RT-F2/R2 (Fig. 3E). Both results suggest the presence of an upregulated allele of PRL2 in sop1-1D, acting as suppressor of prl1-2.  PCR-based amplification of the PRL2 allele present in prl1-2 sop1-1D using primers PRL2-CLF1 and PRL2-CL-R1 did not yield an amplification product whereas no problems were encountered when genomic wild-type DNA was used in the reaction. Small amounts of PCR product were obtained using a non-commercial Paq and primers PRL2-CL-F1 and PRL2-SQ-R1 for amplification of a 2.7kb 5’ fragment and PRL2-SQ-F2 and PRL2-CL-R1 for amplification of a partially overlapping 2.2kb 3’ fragment, however amino acid codon changing mutations were detected in each of four sequenced sections. An attempt to produce a collection of full-length 41  products from twenty independently cloned and restriction nuclease treated 5’ and 3’ fragments failed repeatedly and we changed strategies to achieve complementation.  In an alternative approach, three clones of the Arabidopsis JAtY library were used to transform prl1-2 and prl1-2 sop1-1D mutants (John Innes Centre, http://orders2.genomeenterprise.com/libraries/arabidopsis/jaty). JAtY clone 69M23 covers a 67kb region on Chromosome 3 encompassing PRL2, whereas 79F11 (78kb) and 51K01 (31kb) are adjacent and partially overlapping clones to the left and right, respectively (Fig. 4A). Since experimental data derived from our expression analysis demonstrated upregulated PRL2 transcripts in prl1-2 sop1-1D plants, we proposed that introduction of a second PRL2 copy (JAtY69M23) into the original prl1-2 mutant would result in similar transcript levels and mimic complementation. Consistent with our hypothesis, two of the three JAtY69M23-derived prl1-2 T1 transgenics no longer exhibited mutant phenotypes but wild-type morphology instead (Fig. 4B). The third line displayed a different mutant phenotype (data not shown).Transformation of prl1-2 with JAtY51K01 yielded transgenics with unchanged mutant phenotypes, no transformants were recovered using the largest clone, JAtY79F11 (Fig. 4C). Transformation of wild-type looking prl1-2 sop1-1D plants with any of the three clones resulted in phenotypically unaltered transgenics in all cases (Fig. 4D). The results of our transformation series strongly support PRL2 transcript-level dependent suppression of prl1-2 phenotypes in prl1-2 sop1-1D, as only JAtY69M23-transformed prl1-2 plants exhibited a change in phenotype. In conclusion, the PRL2 allele present in sop1-1D most likely carries a regulatory gain-of-function mutation (prl2-1D), conferring dominant suppression of prl1 mutant phenotypes.  3.3.4 PRL2 and PRL1 exhibit high structural and sequence homology Significant sequence homology between PRL1 and PRL2 proteins has been previously established through hybridization experiments and sequence alignments (Baruah et al., 2009; 42  Nemeth et al., 1998). Both proteins belong to the Transducin/WD40 repeat family, a motif which has been shown to mediate protein-protein interactions (Xu and Min, 2011; Stirnimann et al., 2010). The seven highly conserved WD40 repeats starting at amino acid position 142 of PRL1 and position 135 of PRL2, respectively, make up the majority of the protein structure. In contrast to 89% amino acid identity across this C-terminal region, only 59% identity is shared among the N-terminal amino acids leading up to the previous positions (Fig. 5). A second motif, designated DWD (DDB1 binding WD40) is located within the WD3/4 and WD4/5 repeats of PRL1 and PRL2 (Fig. 5). Recent evidence suggests that a subset of WD40 proteins carrying this motif, among them PRL1, may interact with CUL4-based E3 ligases (Lee et al., 2008). PRL1 localizes to the nucleus using either nuclear localisation signal (NLS) based translocation or potentially through interaction with IMPORTIN ALPHA 3/MOS6 (Kosugi et al., 2009). The high level of sequence conservation between PRL1 and PRL2 proteins accounts as further evidence for functional redundancy between the homologs and is consistent with the ability of prl2-1D to suppress prl12 phenotypes.  3.3.5 Unequal genetic redundancy between PRL1 and PRL2 In contrast to prl1 mutant plants, recessive loss-of-function of PRL2 (prl2-2, Salk_075970) does not lead to morphological changes. To test for redundancy between PRL2 and PRL1, we crossed exonic T-DNA insertion alleles prl1-2 and prl2-2 and identified the prl1-2 prl2-2 double in the F2 generation by PCR-based genotyping. The double mutant can be distinguished from the prl1-2 single mutant in the third week of development since leaves of prl1-2 prl2-2 plants are darker, smaller and rounder (Fig. 6A, B). When a second T-DNA allele, prl2-3 (Salk_133878) was used to generate the prl1-1 prl2-3 double mutant, it also displayed the enhanced phenotype (data not shown). An early flowering phenotype, which is usually observed in prl1-2 single mutants, manifests itself even more pronounced in the double mutant. prl1-2 prl2-2 plants initiate bolting with 7.4 ± 0.5 leaves present, compared to 12.8 ± 1.8 leaves for prl1-1 single 43  mutants and 18.7 ± 1.9 leaves in wild-type plants (Fig. 6C). This trend is less obvious when considering average root lengths, however a slight reduction in lengths was recorded compared to already strongly reduced prl1-2 roots (Fig. 6D). The enhanced phenotypes observed in the double mutant suggest unequal genetic redundancy between PRL1 and PRL2.  3.3.6 prl1-2 prl2-2 mutants are impaired in basal and R-protein mediated resistance Consistent with the obtained morphological data, infection assays reveal no differences in resistance between prl2 loss-of-function and wild-type plants when using a low dose of virulent P.s.m. 4326. Substantially impaired basal defence signalling however was observed for prl1-1 prl2-1 although not surpassing bacteria titer of prl1-2 plants (Fig. 6E). The infection series with virulent oomycete H.a. NOCO2 also indicates less functional low-level defence in the double mutant (Fig. 6F).  For testing R protein mediated resistance, rosette leaves were infiltrated with avirulent Pseudomonas syringae pathovar tomatoe (P.s.t.) DC3000 expressing avrRPS4. This effector is recognized by the TIR-NB-LRR protein RESISTANCE TO PSEUDOMONAS SYRINGAE 4 which triggers effective defence in wild-type (Col-0) and prl2-2 mutant plants. Modification of the responsible signal relay however allows for ten times more growth of the pathogen in prl1-2 and prl1-2 prl2-2 mutants, when titers were calculated at three days past exposure (Fig. 6G). Using oomycete H.a. EMWA1 conidiospores in spray-inoculation experiments, some impaired defence was detected in prl1-2 prl2-2 but prl1-2 plants still sustained more growth of the oomycete (Fig. 6H).  44  3.3.7 prl2-1D single mutants do not show enhanced resistance to pathogens At the genomic level, PRL2 is larger than PRL1 (4.3kb and 3.9kb, respectively) due to increased intron size and longer non-translated regions. The prl2-1D mutation affects the 12th position in the 69bp long 5’ untranslated region of PRL2 thus identifying an important segment for transcript level regulation. We attempted to compare 5’UTR sequences between the homologs but the regions are too different to allow an alignment. The 12th position of the shorter, 47bp measuring 5’UTR of PRL1 is also occupied by a cytosine, both adjacent nucleic acids are however different (data not shown).  We generated the prl2-1D single mutant to investigate whether increased levels of PRL2 transcript combined with wild-type PRL1 expression rates would result in an observable mutant phenotype. The single mutant was created through crossing of prl1-2 prl2-1D with Col-0 and identified in the F2 generation using PCR-based genotyping. Morphologically, prl2-1D mutants resemble a wild-type plant aside from a late flowering phenotype which we have also noted in prl1-2 prl2-1D double mutants. Both mutant types initiate flowering with at least 20 rosette leaves present in contrast to prl1-2 and wild-type plants which flower on average at the 11- and 15-leave stage, respectively (Fig. 7A,B).  Inoculation with virulent bacterial and oomycete pathogens detected no change in basal resistance levels in prl2-1D compared to pathogen growth sustained by prl1-2 prl2-1D plants and by the Col-0 control (Fig. 7C,D). Similarly, colonization by avirulent strains was limited to wild-type levels in prl2-1D when evaluated after three days. P.s.t. DC3000 expressing avrRPS4 and P.s.t. DC3000 expressing avrPphB are detected in a plants cytoplasm by TIR-type and CCtype R proteins, respectively (Fig. 7E, F). When challenged with avirulent H.a. EMWA1 (detected by TIR-NB-LRR immune receptor RPP4), we also did not detect changes in 45  resistance (Fig. 7G). These results suggest that both basal and ETI immunity are unaffected in prl2-1D plants which express PRL2 at significantly increased levels. It is thus unlikely, that the function of PRL1/PRL2 in resistance signalling can be enhanced through a dosage-dependent mechanism such as overexpression of PRL2.  3.4  Discussion  PRL1 is part of the MAC, a multi-protein complex with a role in plant defence signalling. It is suggested, that the WD40 protein contributes substantially to a plants defence output since loss of PRL1 results in higher pathogen colonization than observed in other MAC mutants i.e. mos4, Atcdc5 and mac3a mac3b (Monaghan et al., 2009; Palma et al., 2007). With the goal of identifying new defence components functioning in PRL1 - and MAC associated signal relay, we have carried out a genetic suppressor screen in the prl1 mutant background. Investigation of the dominant suppressor prl2-1D suggests that not only well-known PRL1 but also the PRL2 homolog are involved in the regulation of plant immunity, sugar sensitivity and morphogenesis.  Important aspects of cellular functions such as transcription and ubiquitination are mediated with the help of specialized protein sequences such as the zinc-finger motif or the Ring-finger domain, respectively. Notably, these domains are among the most abundant in eukaryotic proteomes whereas modules that have been implicated in signalling events, such as the interactor domains SH2 (src homology 2), SH3 and PDZ (postsynaptic density 95/ discs large/zonula occludens-1) are not as common and less conserved in importance across kingdoms (Stirnimann et al., 2010). For example, the interactor domain SH2 connects an extensive network of phosphorylated tyrosine-containing proteins in animals whereas only two Arabidopsis proteins feature a predicted homologous motif (Pawson, 2007; Williams and Zvelebil, 2004). More recently, the importance of the WD40 family is emerging which is the most utilized interaction module in baker’s yeast and also makes up approximately 1% and 0.8% of 46  proteins in a limited human and Arabidopsis proteome analysis, respectively (Stirnimann et al., 2010)  The majority of the PRL2 and PRL1 protein structure, about 70%, is taken up by seven WD40 repeat sequences whereas the remaining C-terminal region does not seem to comprise another motif. The encoded 7-fold WD40 propeller is a highly symmetrical structure allowing interaction to occur on all sides of the surface including the top, bottom and circumference (Xu and Min, 2011). This binding flexibility is certain to play a role in modulating the multitude of regulatory activities established for PRL1 (Flores-Perez et al., 2010; Baruah et al., 2009; Lee et al., 2008; Palma et al., 2007; Nemeth et al., 1998). The WD40 structure is approximately 90% conserved between the PRL2 and PRL1 homologs thus likely capable of a similar range of interactions and assumed to be responsible for re-established wild-type phenotypes in the prl1-2 prl2-1D mutant.  So far, WD40 proteins have not been shown to exhibit enzymatic activity but rather are suggested to function as adapter components in signal relay (Stirnimann et al., 2010). Increased PRL2 mRNA levels in the prl2-1D single mutant did not result in a detectable mutant phenotype, in line with a presumed structural role of PRL2 and PRL1. Through reversible or stable association with pathway controlling elements, adapter proteins often provide essential platforms for regulatory interaction. Such a role could explain the enhanced susceptibility displayed by prl1-2 and prl1-2 prl2-1 mutants compared to other MAC mutants. The presence of yet another predicted structural protein in the MAC core complex, MOS4, emphasizes the importance of stable interplay for functional immunity signalling.  Phenotypes of prl1-2 and prl1-2 prl2-2 mutants are not limited to defects in immunity. It is conceivable, that PRL1 and PRL2 might be only transiently associated with MAC components for defence purposes while also mediating interactions in other pathways. A presumed function as a signalling-enabling adapter could explain involvement of PRL1 in diverse signalling 47  branches such as sugar, light, stress, resistance and hormone responses (Flores-Perez et al., 2010; Baruah et al., 2009; Lee et al., 2008; Palma et al., 2007; Nemeth et al., 1998). Influence of PRL1/PRL2 over additional pathways independent from the MAC is also supported by cloning of suppressor of prl1, 2Dominant, (sop2-1D), which specifically reverses prl1phenotypes however does not affect mutant atcdc5 or mos4 signalling (Weihmann et al., unpublished results).  In contrast to the highly conserved protein-coding segments, regulatory sequences of PRL2 and PRL1 have diversified significantly. PRL2 transcript levels are far lower than those of PRL1 in wild-type Arabidopsis plants potentially as a result of PRL2 down regulation. Despite the differences, a single polymorphism at the 12 th position of the PRL2 5’ untranslated region (5’UTR) substantially increases mRNA levels suggesting that an important regulatory motif is present in this region. Single nucleotide polymorphisms (SNP) in 5’UTRs have been studied in some detail in human genes since such changes often correlate with disease phenotypes (Chatterjee and Pal, 2009). For example, six different SNPs were found in alleles of ANKRD26 (ankirin repeat domain 26) and are associated with thrombocytepemia, a condition of abnormally low amounts of platelets. Reporter gene fusion constructs consisting of mutated 5’UTRs and the luciferase ORF demonstrated significant overexpression for all tested constructs (Pippucci et al., 2011). Similarly, a single base substitution in the 5’ UTR of TGFβ3 (transforming growth factor-beta3) causes more than two-fold increased expression in a TGFβ3 5’UTR-luciferase assay. This mutant allele of TGFβ3 contributes to the development of a specific type of myocardial disease in young adults (Beffagna et al., 2005). The upstream untranslated region of TGFβ3 contains 11 ATG sequences which potentially initiate several upstream open reading frames (uORFs) (Beffagna et al., 2005). uORFs can affect translation of the main open reading frame, a regulatory mechanism that is also been present in yeast and plants (Calvo et al., 2009; Tran et al., 2008; Zhang and Dietrich, 2005). Neither PRL1 nor PRL2 UTRs however contain any additional ATG sequences upstream of the inferred translational 48  start codon and there are no strong indications of alternative transcripts (J. Robertson, personal communication). The rate of translation may also depend on the length of a given UTRs and potential secondary structures among other mechanisms. Finally, we cannot rule out that the polymorphism in the PRL2 gene affects the rate of transcription rather than translation and stability of produced RNA molecules.  An often cited aspect for the decision of positioning Arabidopsis thaliana en route to the successful model it is today has been the compact genome size and low amount of repetitive sequences compared to the complex genomes of related crop plants. ollowing the Arabidopsis genome release and extensive in silico analysis, it became clear that there had also been several large-scale duplication events in the adopted model plant, since approximately 80% of the identified genes are present in two or more copies (Blanc and Wolfe, 2004). Examples of largely maintained redundancy such as for the two loci encoding E3 ubiquitin ligases subunits ATCUL3A/3A have been documented, however predicted outcomes for at least one copy of a duplicated gene also include diversification of function, expression patterns or even a fate as non-functional pseudogene (Briggs et al., 2006; Figueroa et al., 2005). In the MAC, U-BOX proteins MAC3A and MAC3B function redundantly in plant immunity whereas the relationships between the three MAC5 loci are more complex, displaying unequal genetic redundancy between MAC5A and MAC5B-encoded proteins and non-reduncancy between MAC5C and MAC5A/MAC5B (Monaghan et al., 2010; Monaghan et al., 2009). Absence of a mutant phenotype in prl2-2 plants and an enhanced phenotype displayed by prl1-2 prl2-2 mutants are hallmarks of unequal genetic redundancy caused by substantially reduced expression of one of the duplicates (Briggs et al., 2006). However, PRL2 appears still as functional as PRL1.  The fate of both the ancestral and a daughter gene is not pre-assigned following a duplication event. Duplicated genes can be the source of novel gene functions (neofunctionalization) or may result in an inactive pseudogene state for one of the copies (nonfunctionalization) (Ohta, 49  2000). Subfunctionalization on the other hand, is an evolutionary mechanism believed to be contributing to the preservation of duplicated genes. In this process, both members of a gene pair experience degenerative mutations that affect expression patterns and level of activities (Lynch and Force, 2000). We did not detect novel properties of the PRL2 protein in the overexpressing sop2-1D mutant which argues against neofunctionalization. According to the AtGenExpress platform, PRL2 expression ranks consistently five times lower than PRL1 throughout developmental stages, with the exception of pollen tissue. In this floral organ, PRL1 expression drops whereas PRL2 expression reaches levels five times higher than PRL1. Such inverted expression patterns could be seen as evidence towards subfunctionalization, i.e. developing tissue specificity. Mutations in the regulatory promoter region likely are responsible for the already strongly attenuated expression of PRL2 and might ultimately lead to full pseudogenization of the homolog (Yang et al., 2011; Adams and Wendel, 2005).  50  A A  prl1-2  WT  prl1-2 sop1-1D  C  B  30 Number of rosette leaves at bolting  Average root length [mm]  40 30 20 10  **  * 20  ** 10 0  D  P.s.m ES4326 Day 0  E  Day 3  8  **  5  6  H.a. EMWA1 10  ** spores (x10 )/g  4 2 0  6 4 2 W T pr l12 so p1 -1 D  2 s prWT op l1-2 11D pr l1-  pr l1  -2 p WT so rl1 p1 -2 -1 D  0  pr l12  log cfu/cm  2  8  W T pr p l1rl1 2s -2 op 11D  W T pr pr l1l 1 2s -2 op 11D  0  Figure 2. sop1-1D suppresses prl1 related phenotypes (A) Morphology of soil-grown WT (Col-0), prl1-2 and prl1-2 sop1-1D. (B) Root length analysis of one week old seedlings of indicated phenotypes, results represent an average of ten seedlings each ±SD. (C) Flowering time analysis: rosette leaves of ten plants per genotype WT (Col-0), prl1-2 and prl1-2 sop1-1D were counted when emerging flower measured 1cm in height. Values represent averages ±SD. (D) Infection experiments with P.s.m. ES4326 (OD600= 0.0001) using five-week-old soil-grown plants of indicated phenotypes. Bacterial titer was quantified at 0 and 3 days post inoculation, values represent average of six replicates per genotype ±SD. (E) Soil-grown, 2.5 week old plants of indicated genotypes were spray inoculated with 100,000 spores/ml of H.a. EMWA1 and colonisation quantified after nine days. Values represent averages ±SD of two replicates with 15 plants each. For (B) through (E), experiments were repeated at least three times with similar results. Statistical significance compared to Col-0 control was calculated using Student’s t-test: *P < 0.001 and **P < 0.0001 for all graphs. cfu, colony forming units.  51  A  C  unscaled expression  2.5 2.0 1.5 1.0 0.5  -2 pr l2  1D so p1 -  so p1 -1 D  -2 pr l1  -2  pr l1  W T  0  D  -2 pr l2  so p1 -  pr l1  -2  so p1 -1 D  1D  -2 pr l1  W T  B  Figure 3. Map-based cloning of sop1-1D (A) Map position of sop1-1D; indicated are recombinants and sequence-anchored positions of flanking markers and BAC clones. A mutation (*) was identified in At3g16650/PRL2. (B) Sequence analysis reveals a point mutation in the PRL2 5'UTR of prl1-2 sop1-1D. (C) Quantification of real-time RT-PCR data using exon-specific PRL2 primer on c-DNA obtained from indicated genotypes of tissue series #1. Values represent averages of two experimental replicates. (D) Semi-quantitative RT-PCR of tissue series described in C).  52  A  C Chromosome 3 5.7  5.6 JAtY79F11  (Mb)  JAtY51K01  library clones  JAtY69M23  prl1-2 (mutant phenotype)  prl1-2 sop1-1D (WT phenotype)  79F11  69M23  51K01  n.d.  2 WT / 3 total  6 mutant / 6 total  4 WT / 4 total  5 WT / 5 total  5 WT / 5 total  At3g16650/ PRL2  B  prl1-2 sop1-1D  D prl1-2  prl1-2 sop1-1D with JAtY69M23, T1  prl1-2 with prl1-2 with JAtY69M23, JAtY69M23, T1, line #1 T1, line #2  Figure 4. Overexpression of PRL2 is able to complement prl1 mutant defects (A) Arrangement and sequence-anchored positions of JAtY clones 79F11, 69M23 and 51K01. The PRL2 gene is only covered by JAtY69M23. (B) Morphology of prl1-2 sop1-1D, prl1-2 and two transgenic lines obtained through introduction of JAtY69M23, which harbours PRL2, into prl1-2. (C) Summary of JAtY transformations; Indicated are JAtY clones, genotypes of transformed plants as well as number and phenotypes of obtained transgenic plants. (D) Morphology of transgenic plants obtained through transformation of JAtY69M23 into prl1-2 sop1-1D.  53  A bipartite NLS  PRL1 PRL2  1 MPAPTTEIEPIEAQSLKKLSLKSLKRSLELFSPVHGQFPPPDPEAKQIRLSHKMKVAFGG 1 MTMIALNRE-VETQSLKKLSLKSVRRAREIFSPVHGQFPQPDPESKRIRLCHKIQVAFGG monopartite NLS  PRL1 PRL2  61 VEPVVSQPPRQPDRINEQPGPSNALSLAAPEGSKSTQKGATESAIVVGPTLLRPILPKGL 60 VEP-ASKPTRIADHNSEKTAPLKALALPGPKGSKELRKSATEKALVVGPTLP----PRDL  PRL1 PRL2  121 NYTGSSGKSTTIIPANVSSYQRNLSTAALMERIPSRWPRPEWHAPWKNYRVIQGHLGWVR 115 NNTGNPGKSTAILPAPGSFSERNLSTAALMERMPSRWPRPEWHAPWKNYRVLQGHLGWVR  WD1  WD2  PRL1 PRL2  181 SVAFDPSNEWFCTGSADRTIKIWDVATGVLKLTLTGHIEQVRGLAVSNRHTYMFSAGDDK 175 SVAFDPSNEWFCTGSADRTIKIWDVATGVLKLTLTGHIGQVRGLAVSNRHTYMFSAGDDK WD3  PRL1 PRL2  241 QVKCWDLEQNKVIRSYHGHLSGVYCLALHPTLDVLLTGGRDSVCRVWDIRTKMQIFALSG 235 QVKCWDLEQNKVIRSYHGHLHGVYCLALHPTLDVVLTGGRDSVCRVWDIRTKMQIFVLPDWD box 1  WD4  PRL1 PRL2  DWD box 2  PRL1 PRL2  WD5  301 HDNTVCSVFTRPTDPQVVTGSHDTTIKFWDLRYGKTMSTLTHHKKSVRAMTLHPKENAFA 294 HDSDVFSVLARPTDPQVITGSHDSTIKFWDLRYGKSMATITNHKKTVRAMALHPKENDFV WD6  361 SASADNTKKFSLPKGEFCHNMLSQQKTIINAMAVNEDGVMVTGGDNGSIWFWDWKSGHSF 354 SASADNIKKFSLPKGEFCHNMLSLQRDIINAVAVNEDGVMVTGGDKGGLWFWDWKSGHNF WD7  PRL1 PRL2  421 QQSETIVQPGSLESEAGIYAACYDNTGSRLVTCEADKTIKMWKEDENATPETHPINFKPP 414 QRAETIVQPGSLESEAGIYAACYDQTGSRLVTCEGDKTIKMWKEDEDATPETHPLNFKPP  PRL1 PRL2  481 KEIRRF 474 KEIRRF  Figure 5. Alignment of PRL1 and PRL2 protein sequences Dark shaded areas denote identical amino acids, light grey areas indicate similar amino acids. Predicted NLS according to cNLS mapper shown for PRL1 above and for PRL2 below the alignment (Kosugi et al., 2009). WD40 repeats 1-7 (adapted from Nemeth et al., 1998 and Baruah et al., 2009) and DWD boxes 1 and 2 (Lee et al., 2008) are indicated for both sequences. NLS, nuclear localization signal. DWD, DDB1 binding WD40.  54  **  0  1kb  Salk_133878  WT  **  10  E  prl1-2  6 4 2  6  Day 0  H  Day 3  ** **  4 2 W pr T l1 pr l1- prl -2 2 p 2-2 rl2 -2 W pr T l1 pr l1- prl -2 2 p 2-2 r l2 -2  0  H.a. NOCO2 30 20  ** ** **  10  H.a. EMWA1 10  spores (x10 )/ g  log cfu/cm  2  8  **  8  **  6 4 2 0 W pr T l 1 pr l1- prl -2 2 p 2-2 rl2 -2  P.s.t. avrRps4  5  G  **  0  Co pr l-0 l1 pr l1- pr -2 2 p l2rl2 2 -2 Co p r l- 0 l1 pr l1- prl -2 2 p 2-2 rl2 -2  prl1-2 prl2-2  F  5  2  log cfu/cm  ** **  0 prl2-2  10 0  P.s.m. ES4326 Day 0 Day 3  8  20  Co pr l-0 l1 pr l1- prl2-2 2 p -2 r l2 -2  *  Stop  20  Co l-0 pr l1p 2 pr l1- rl2-2 2p rl2 -2  PRL2  30  Co lpr 0 l12 pr prl l1- 22p 2 rl2 -2  Salk_075970  D 30  spores (x10 )/g  1kb  Salk_008466  B  C  Stop  Average root length [mm]  PRL1  ATG  Number of rosette leaves at bolting  A  Figure 6. Enhanced phenotypes in prl1 prl2 double mutants compared to single mutants (A) Gene structure of PRL1 and PRL2. Indicated are positions of Salk lines S_008466 (prl1-2), S_075970 (prl2-2) and S_13378 (prl2-3). The prl2-1D mutation (*) is located in the first exon. (B) Morphology of soilgrown WT (Col-0), prl1-2, prl2-2 and prl1-2 prl2-2. (C) Flowering time analysis of WT, prl1-2, prl2-2 and prl1-2 prl2-2. Rosette leaves of ten plants per genotype where counted when emerging flower measured 1cm in height. Values represent averages ±SD. (D) Root length analysis of indicated genotypes. Results represent an average of ten plate-grown seedlings for each genotype ±SD. (E) Bacterial infection of indicated phenotypes with virulent P.s.m. ES4326 (OD600= 0.0001) using five week old soil-grown plants. Bacterial titer quantification at 0 and 3 dpi, averages represent six replicates ±SD. (F) Evaluation of disease susceptibility of indicated genotypes towards virulent oomycete H.a. NOCO2 (50,000 spores/ml), values represent averages of two replicates of 15 plants ±SD. (G) Inoculation of indicated genotypes with avirulent P.s.t. avrRPS4 (OD600= 0.002) using five week old soil-grown plants. Bacterial titer quantification at 0 and 3 dpi, averages represent six replicates ±SD. (H) Evaluation of disease susceptibility of indicated genotypes towards avirulent oomycete H.a. EMWA1 (200,000 spores/ml), values represent averages of two replicates of 15 plants ±SD. For (C) through (H), experiments were repeated at least three times with similar results. Statistical significance compared to Col-0 control was calculated using Student’s t-test: **P < 0.0001 for all graphs. cfu, colony forming units.  55  0  0  Day 3  10  **  8  **  5  4  2  6 4  pr l1-  W T 2 p prl1 rl2 -2 pr -1D l21D  0  0  pr l1- p WT 2 p rl1 rl2 -2 pr -1D l21D  pr l1- p WT 2 p rl1 rl2 -2 pr -1D l21D  W p T 2 p rl1rl2 2 pr -1D l21D  H.a. EMWA1  2  0  pr l1-  Day 0  2  4 2  0  6  G  spores (x10 )/g  10  6  W 2 p prl1 T rl2 -2 pr -1D l21D  P.s.t. avrPphB  W pr l1- p T 2 p rl1 rl1 -2 pr -1D l21D  **  Day 3  log cfu/cm  20  2  ** **  Day 0  *  4 2  ** log cfu/cm  Nº of leaves at bolting  8  Day 3  6  10  P.s.m. ES4326 30  2  20  F  C  B  **  30  prl2-1D  Day 0  pr l1-  prl1-2 prl2-1D  P.s.t. avrRps4 8  W pr l1- p T 2 p rl1 rl2 -2 pr -1D l21D  5  spores (x10 )/g  40  E  W 2 p prl1 T rl2 -2 pr -1D l21D  H.a. NOCO2  pr l1-  D  prl1-2  log cfu/cm  WT  W pr l1- pr T 2 p l1rl2 2 pr -1D l21D  A  Figure 7. Basal and R protein mediated resistance is unaffected in prl2-1D (A) Morphology of five-week-old soil-grown Col-0 (WT), prl1-2, pr1-2 prl2-1D and prl2-1D. (B) Flowering time analysis of indicated genotypes. Rosette leaves of ten plants per genotype where counted when emerging flower measured 1cm in height. (C) Infection assay of indicated genotypes with five-week-old plants using a low dose (OD600= 0.0001) of P.s.m. ES4326 and quantification of bacterial titer at 0 and 3 dpi. Values represent six replicates ±SD. (D) Evaluation of resistance towards virulent oomycete H.a. NOCO2 of indicated genotypes. Spray-inoculation of 2.5-week-old plants with 50.000 spores/ml followed by conidiospore count after 8 days. Values represent two replicates of 15 plants ±SD. (E) and (F) Evaluation of disease resistance towards avirulent bacterial pathogens. Injection of a high dose (OD600= 0.002) of P.s.t. DC3000 expressing avrRPS4 or avrPphB into leaves of five-week-old plants of indicated phenotypes showed wild-type resistance in prl2-1D. Bacterial titer ere quantified at 0 and 3 dpi. Values represent six replicates ±SD. (G) Spray-inoculation of 2.5-week-old plants of indicated genotypes with 150.000 spores/ml of avirulent oomycete H.a. EMWA1 followed by conidiospore count after 8 days, values represent averages of two replicates with 15 plants. Statistical significance compared to Col-0 control was calculated using Student’s t-test: *P < 0.003 and **P < 0.0001 for all graphs. dpi, days post inoculation.  56  4  A dominant mutation in an uncharacterized gene  identifies a component of PRL1 signalling specific to the plant kingdom 4.1  Introduction  In plants, perception of pathogens is facilitated by receptors, which monitor the extracellular environment and activities in the cytosol. The first line in active defence is provoked once conserved epitopes of bacterial and fungal pathogens such as flagellin, lipopolysaccharides, chitin or other pathogen associated molecular patterns (PAMPs) have been detected by pattern recognition receptors at the cell surface (Willmann et al., 2011; Newman et al., 2007; GomezGomez and Boller, 2000). PAMP-triggered immunity (PTI) is effective against a broad range of pathogens; early activities include ion fluxes across the plasma membrane as well as mobilisation of reactive oxygen species and signalling via the mitogen-activated protein kinase (MAPK) network which helps regulate the PTI defence output through activation of WRKY-type transcription factors (Petersen et al., 2010; Nicaise et al., 2009; Qiu et al., 2008; Asai et al., 2002). Successful pathogens are nonetheless able to deliver effector molecules into the host cell, which often function to diminish or suppress PTI, thus allowing virulent pathogen growth. In incompatible interactions however, the presence or activity of effectors is detected by resistance (R) proteins which are intracellular immunity receptors. Activation of R protein complexes initiates effector-triggered immunity (ETI), an amplified form of PTI which confers resistance against specialized pathogens.  R protein induced ETI signalling in response to biotrophic pathogens is usually first routed through lipase-like proteins ENHANCED SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN DEFICIENT 4 (PAD4) which control salicylic acid –dependent responses (Venugopal et al., 57  2009; Wiermer et al., 2005; Parker et al., 1996). Some CC-type R proteins also signal though NONRACE-SPECIFIC DISEASE RESISTANCE (NDR1), a transmembrane protein with homology to integrins (Knepper et al., 2011; Aarts et al., 1998). Pathogen-induced SA production and accumulation are facilitated by the isochorismate synthase SID2 (SA INDUCTION DEFICIENT2) and MATE transport family protein ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) and supported by the ALD1 protein (AGD2-LIKE DEFENCE RESPONSE) (Song et al., 2004; Nawrath et al., 2002; Wildermuth et al., 2001) SENESCENCE ASSOCIATED GENE 101 (SAG 101), a third lipase –like protein forms a ternary complex with EDS1 and PAD4 in the nucleus with a role in SA mediated defence (Zhu et al., 2011). Redox changes in the cytoplasm lead to monomerization and relocation of usually inactive complexed multimers of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) (Tada et al., 2008; Mou et al., 2003). In the nucleus, NPR1 binds TGA family transcription factors which orchestrate defence-related transcription , likely supported by WRKY proteins (Kesarwani et al., 2007; Eulgem, 2006; Wang et al., 2006; Zhang et al., 2003). Mutants deficient in SA production or acculmulation, or NPR1 function, have demonstrated abilities to build up partial defences thus identifying signalling routes independent of both regulators (Lu, 2009; Lee et al., 2007; Palma et al., 2007; Bartsch et al., 2006a; Bowling et al., 1997)  Resistance pathways have been further examined through genetic dissection of snc1 (suppressor of npr1-1, constitutive 1) signalling, a dwarf mutant encoding a constitutively activated predicted R protein (Zhang et al., 2003). New defence elements were identified through cloning of mos (modifier of snc1) mutations which partially or fully restored wild-type phenotypes in snc1 mos suppressor mutants (Monaghan et al., 2010). For example, the nuclear transport machinery was found to be required for plant resistance since MOS3, MOS6 , MOS7 and MOS11-encoded proteins function in messenger RNA export as well as both import and export of nucleus-targeted proteins (Germain et al., 2010; Monaghan et al., 2010). Posttranslational farnesylation and ubiquitination mechanisms do also play a role in defence 58  (MOS5/UBA1 and MOS8/ERA1) (Cheng et al., 2011; Goritschnig et al., 2008; Goritschnig et al., 2007).  Characterization of MOS4 revealed association with ~20 proteins, either stable or transiently organized in a complex termed MAC (for MOS4-associated complex) (Monaghan et al., 2009; Palma et al., 2007). Loss-of-function mutations in predicted MAC structural elements MOS4, PRL1 (PLEIOTROPIC REGULATORY LOCUS 1) or putative transcription factor CELL DIVISION CYCLE 5 (AtCDC5), result in increased colonization by both virulent and avirulent pathogens (Palma et al., 2007). Similar susceptibility was also demonstrated in mac3a mac3b mutants which lack two MAC U-box proteins with homology to the yeast and human E3 ubiquitin ligase Prp19 (Monaghan et al., 2009). Defence defects have been established for all tested MAC proteins although plants with mutations in PRL1 tested most impaired. With the goal of identifying potential targets of this specific MAC member and aiming to clarify tasks of the complex in plant defence, we based a suppressor screen on a mutant allele of PRL1. Our screen resulted in a range of suppressors, the first of which was described recently (Weihmann et al., submitted). Here we describe cloning and characterization of sop2-1D, a dominant mutant obtained from the prl1 suppressor screen.  4.2  Material and methods  4.2.1 Plant material and growth Wild-type Arabidopsis ecotypes Columbia (Col-0), Landsberg erecta (Ler), Wassilewskija (Ws) and derived mutants were grown on soil in a 16h light / 8h dark regime. T-DNA mutants prl1-2 (Salk_008466), sop2-2 (Salk_058710) and sop2h-1 (Salk_121713c) were obtained from the Arabidopsis Biological Research Centre, atcdc5-2 (GABI_278B09) was obtained from the MaxPlanck-Institute for Plant Breeding Research. All lines were genotyped by PCR, using insertion 59  flanking oligonucleotides PRL1-Salk-NF (5'-GATGAAAGTTGCGTTTGGAG-'3) and PRL1-NR-A (5’-ACTACCTACACTACCTAGAGC-‘3) to identify prl1-2 mutants, Salk_058710-F (5'AGCCACATCTCTTGCTGTTG-'3) and Salk_058710-R (5'-TGAAGCGAGAAGCGGTAACT-'3) for identification of sop2-2 mutants, Salk_121713c-F (5'-CTAGGACTCCCATCTGCACTA-'3) and Salk_121713c-R (5'-AATGGCATGTCTCAAGTTGG-'3) for identification of sop2h-1 mutants.  4.2.2 Plant pathogens and infection assays Bacterial pathogens Pseudomonas syringae pathovar maculicola (P.s.m.) ES4326 and pathovar tomato (P.s.t.) are hemi-biotrophic bacterial pathogens responsible for leaf spot and bacterial speck disease on Arabidopsis, respectively. For propagation, bacteria were cultivated on LB plates containing 50 mg/ml Streptomycin (P.s.m. ES4326) or 25 mg/ml Rifampicin and 50 mg/ml Kanamycin (P.s.t. avrPphB and avrRps4). For inoculation, bacteria were grown at 28-30ºC in liquid LB to high densities, cross-inoculated to fresh media and allowed to reach OD 600 = 0.2. Using a needless small plastic syringe lightly pressed against an Arabidopsis leave underside, bacterial solution is injected into the apoplast. A low dose (OD600= 0.0001), referred to as enhanced disease susceptibility (EDS) dose, was used in virulent (P.s.m. 4326) infections. A high dose (OD600= 0.002) was employed with avirulent P.s.t. avrPphB and P.s.t. avrRps4. On the first day and three days after inoculation, leave discs of 0.32cm 2 were cut with a standard paper hole-punch, samples were homogenized in 10mM MgCl 2 and a series of six dilutions was plated. The bacterial titer was measured after two days of incubation at 28ºC.  Hyaloperonospora arabidopsidis (formerly Peronospora parasitica and Hyaloperonospora parasitica) (Holub, 2008; Slusarenko and Schlaich, 2003) is a biotrophic oomycete pathogen which causes downy mildew on Arabidopsis. It is an obligate pathogen which has to be propagated on susceptible Arabidopsis ecotypes usually completing a life cycle in one week at 60  16ºC and > 50% humidity (McDowell et al., 2011). The susceptible Arabidopsis ecotype Col-0 was used to propagate H.a. NOCO2, H.a. EMWA1 was grown on susceptible Ws plants. For infection assays, 2.5-week-old plants were spray-inoculated using either a low dose (50,000 spores/ml) to determine resistance defects in a compatible interaction, or a high dose (100,000 spores/ml) for evaluation of incompatible interactions. After 7-10 days, plants were harvested in a 50 ml Falcon tube containing up to 5 ml water, conidiospores were released using a vortex for 10 seconds and quantified using a hemocytometer.  4.2.3 Positional cloning Molecular markers used for map-based cloning are PCR based and detect either length (InDel) or single nucleotide polymorphisms (SNP) between Col-0 and Ler (Monsanto Arabidopsis Ler sequence available at TAIR: [http://www.arabidopsis.org/browse/Cereon/index.jsp]).ecotypes. When employing InDel markers, one set of primers is used for both ecotypes and amplified fragments are visualized on 1-2% agarose gels. For SNP markers, two ecotype specific forward primers and one common reverse primer were designed. The forward primers differ in the last two positions with the ultimate nucleotide complementary to the respective ecotype polymorphism, and the penultimate chosen to stabilize specific binding (Bui and Liu, 2009). The primer combinations are used in alternating reactions on genotypes and fragments are analysed on 1% agarose gels. For primer sequences, see Appendix 2.  4.2.4 Single and double mutant construction For creation of the sop2-1D single mutant, homozygous prl1-2 sop2-1D mutants were crossed with Col-0 wild-type plants and the single was identified in the F2 generation using allele specific primers P20A4-M-F (5'-AATGGTTTTCTATCAGGCAT-'3) and P20A4-WT-R2 (5'GAGCAGACCTGTTTCTTAGTCC-'3). Double mutants prl1-2 sop2-2, prl1-2 sop2h-1, sop2-2 61  mos4-1 and sop2-2 atcdc5-2 were generated through crossing of respective single mutants. In all cases, double mutants were identified in the F2 by PCR-based genotyping.  4.2.5 Phylogenetic analysis Protein sequences were obtained from NCBI and PLAZA (Sayers et al., 2011; Proost et al., 2009). For full gene ID’s, see Appendix 4. The 31 plant protein sequences were aligned with MAFFT v.6 using default settings (Katoh et al., 2002). Sequences were edited in SeaView (Gouy et al., 2010). We then used the ProtTest server (http://darwin.uvigo.es/software/prottest_server.html) to determine the best model of protein evolution for our sequences (Abascal et al., 2005).The model chosen according to the Akaike Information Criterion was the JTT model (Jones et al., 1992). Thus, we inferred a maximum likelihood tree under this model using RAxML (Stamatakis et al., 2005). Using RAxML’s rapid bootstrapping method, we performed 500 bootstrap replicates to establish significance for interior branch points (Stamatakis et al., 2008).  4.2.6 Localisation of SOP2-GFP Using primers At2g40638-CL-F (5’-CGGGGTACCTGTTTTATCGCGGGTTTATGTG-3’) and At2g40630-CL-GFP-R (5’-CGCGGATCCATGAAGCCGCCGCCTACCG-3') we amplified a 3.5kb genomic fragment encompassing the SOP2 open reading frame and introduced KpnI and BamHI restriction sites on the 5’ and 3’ ends, respectively, The digested fragment was cloned into a modified pGreen vector just upstream of a GFP open reading frame. Through sequencing of a SOP2-GFP fusion clone we confirmed in- frame insertion and absence of mutations. The construct was transformed into prl1-2 sop2-1D mutants and transgenic plants identified through BASTA® selection. Fluorescence from propidium iodide-stained tissue of BASTA® resistant T2 progeny complementing the sop2 phenotypes was analysed using a Nikon Eclipse 80i confocal 62  laser-scanning microscope (Lucas et al., 2006). Images of GFP expression used the Zeiss (LSM5 Pascal, Germany) confocal microscope as well as the Nikon Eclipse 80i and s using 40x and 60x objective and laser lines, 488 nm and 543nm.  4.2.7 Quantification of SOP2 mRNA levels Seeds of the genotypes prl1-2, atcdc5-2, mos4-1, mac3a mac3b, mac5a and Col-0 wild-type were vernalized for 7 days and plated on 0.5 MS containing 100 mg/ml Ampicillin. The plates were incubated in a growth chamber for ten days using a 16h light, 8h dark regime. Tissue of 10-day-old seedlings was collected in a 2ml tube containing two glass beads and frozen immediately. RNA was extracted using the Totally RNA kit (Ambion) and Reverse Transcriptase (SuperScript II, Invitrogen) was used to produce c-DNA copies of the transcriptome. Relative amounts of SOP2 cDNA (At2g40630_40-F2: 5’-ATAAGCGGAGAGGTGGTGAG-‘3, At2g4063040-R2: 5’-TGATTACCGTCTTTCCCAAA-‘3) and Tubulin (control) c-DNA (5′ACGTATCGATGTCTATTTCAACG-3′ and 5′-ATATCGTAGAGAGCCTCATTGTCC-3′) present in the collected tissues were quantified using real-time PCR.  4.3  Results  4.3.1 Dominant suppression of prl1 loss-of-function phenotypes by sop2-1D To facilitate a more complete picture of PRL1-related contributions to plant defence, we have carried out a genetic screen based on suppression of mutant prl1- associated disease symptoms and morphological criteria (Palma et al., 2007; Nemeth et al., 1998). One of the obtained mutants harbours a dominant suppressor allele, which we named suppressor of prl1,2 Dominant (sop2-1D). The dominant phenotype was detected after back crossing of a prl1 63  homozygous wild type-like progeny plant of a selfing sop2 line with the parental prl1-2 line. The cross resulted in nine prl1-like and twelve wild-type progeny in the first filial generation, indicative of a segregating dominant mutant allele in the tested sop2 plant (expected 1:1, χ2 = 0.42, P = 0.51). sop2-1D is a complete suppressor of mutant leaf colour and shape in the prl1-2 sop2-1D double mutant and also suppresses characteristically short root length, usually associated with the prl1-2 mutant background (Fig. 8A,B).  To investigate the impact of sop2-1D on immunity signalling in the prl1 loss-of-function background, we employed virulent bacterial pathogen Pseudomonas syringae pathovar maculicola (P.s.m) ES4326. In a compatible interaction between a plant and a virulent pathogen, plant defence responses are outperformed by pathogenic proliferation leading to disease symptoms. In the MAC mutant prl1-2, defence-related signalling is compromised leading to even higher colonization compared to a susceptible wild-type plant. In line with our morphological data, prl1-associated enhanced susceptibility is no longer observed in prl1-2 sop2-1D leaves and instead pathogen growth is restricted to wild-type levels (Fig. 8C). sop2-1D suppression thus also extends to defence phenotypes.  4.3.2 A leucine-for-proline substitution in an uncharacterized protein causes differential interference of prl1-related signalling For positional cloning of sop2-1D, we crossed the prl1-2 sop2-1D double mutant (in the Col-0 background) with Arabidopsis ecotype Landsberg erecta (Ler). From the second filial generation (F2) of the mapping cross, we used 48 prl1-like plants to locate sop2-1D on the bottom arm of chromosome 2. Seeds of F2 plants that were prl1-2 homozygous but sop2-1D heterozygous were collected and marker patterns of 1192 progeny plants used to flank the sop2-1D locus using Insertion/Deletion (InDel) markers T2N18 (15.57Mb) and F16B22 (18.40Mb). Dominant suppression of the recessive prl1 mutation allowed us to use 127 64  recombinants of both mutant and wild-type phenotypes in these mapping steps. Allele configuration at the suppressor locus in wild-type plants was extrapolated from segregation ratios in progeny populations. Next, the candidate region was further narrowed down by means of InDel marker T3G21 (16.86MB) and T3K9 (17.10Mb), for which thirteen recombinants remained. Final flanking of sop2-1D was achieved with three and two recombinants for InDel marker T2P4(2) and single nucleotide polymorphism (SNP) marker T7D17-SNP2, respectively (Fig. 9A). Through sequencing of candidate open reading frames, we identified a base pair change in the third exon of At2g40630 (Fig. 9B). The genomic modification results in substitution of proline with leucine in the encoded predicted protein (Fig. 9C)  At2g40630 is an uncharacterized gene of approximately 2.9Kb, with eight exons encoding a 535 amino acid protein (Arabidopsis Genome Initiative, 2000). To confirm that At2g40630 is SOP2, we transformed prl1-2 sop2-1D plants with a 4kb genomic clone encompassing the wild-type At2g40630 open reading frame and 2.3kb of 5’ and 3’ regulatory and non-translated sequences. Two transgenic plants were recovered, both exhibiting prl1-like features although growing slightly larger and exhibiting less serrated leaves (Fig. 9D). When the homozygous T2 plants were challenged with virulent P.s.m. ES4326, they exhibited prl1-like susceptibility indicating that At2g40630 can complement the mutation in prl1-2 sop2-1D (Fig. 9E).  Since sop2-1D is dominant, we also tested the effect of the sop2-1D allele on prl1-2 plants. When prl1-2 single mutants were transformed with an analogue 4kb clone derived from a sop21D homozygous plant and consisting of the mutant sop2 sequence and 2.3kb of adjacent 5’ and 3’ regions, the resulting six T1 plants showed varying levels of prl1 suppression, one line exhibiting a near wild-type phenotype (Fig. 9F).  Since introduction of the mutant sop2-1D allele into prl1-2 resulted in partial suppression and introduction of genomic SOP2 into prl1-2 sop2-1D lead to partial complementation, a dominant65  negative mechanism can be hypothesized. In the presence of both a wild-type SOP2 protein and an altered sop2-1D gene product (such as in all transgenics), the latter may act antagonistically. Considering the range of intermediate traits in the transgenic lines, this interference could be dosage-dependent.  4.3.3 SOP2 and SOP2h are plant-specific proteins In silico BLAST analysis identified 32% amino acid identity of SOP2 with At5g05240-encoded SOP2h, a homolog likely arisen from a genome duplication event (Tang et al., 2008; Altschul et al., 1997). Sequence homology between the proteins is scored throughout the alignment, however little evidence towards a functional domain could be found. A putative twenty-two amino acid coiled-coil domain starting at position 452 (SOP2) and 443 (SOP2h) respectively, is predicted by MIPS (Fig. 10 (Rattei et al., 2010)). When we searched public data bases, we identified further homologous sequences using the NCBI and PLAZA platforms, the latter being a resource for plant genomes (Proost et al., 2009). Similarity with hypothetical proteins present in plant species ranging from established crop plants to newly sequenced cacao and strawberry were established and relationships evaluated using maximum likelihood analysis (Fig. 11). Whereas SOP2 homologs are present in a variety of plant species, none were found in species outside the plant kingdom. Despite PRL1 conservation in eukaryotes, it appears that SOP2 and SOP2h may have a plant specific function.  4.3.4 SOP2 and SOP2h are not essential for plant defence Arabidopsis transcriptome analysis using the AtGenExpress data set revealed no impact of pathogen exposure, abiotic stress or developmental state on the expression of SOP2 (Appendix 5). To examine potential involvement of SOP2 and SOP2h in plant immunity signalling, we investigated whether plants with mutations in these genes would exhibit compromised 66  resistance. We ordered insertional loss-of-function mutants sop2-2 (Salk_058710) and sop2h-1 (Salk_121713c) and obtained the sop2-1D single mutant through crossing of the prl1-2 sop2-1D double mutant with a Col-0 wild-type plant and subsequent PCR-based genotyping in the F2 generation. Morphologically, neither of the two recessive T-DNA alleles nor the dominant sop21D allele caused any obvious morphological defects in singe mutant analysis (Fig. 12A,B). Similarly, when challenged with bacterial pathogen P.s.m. ES4326, all three single mutants sustained normal levels of bacteria three days after inoculation (Fig. 12C).  Genetic redundancy between homologous proteins is considered responsible for the absence of phenotypes in the majority of Arabidopsis single mutants (Briggs et al., 2006).. To investigate, whether the loss of both homologs would produce a mutant phenotype, we crossed sop2-2 and sop2h-1 single mutants and identified the sop2-2 sop2h-double mutant in the F2 by PCR-based genotyping. Transition to flowering was induced slightly earlier in sop2-2 sop2h-1 plants (data not shown) which is a mild phenotype we observed. The approximately wild-type like double mutant however did not show deficiencies in defence mechanisms after inoculation with P.s.m. ES4326. Bacterial colonization levels were similar to those reached in sop2-2, sop2h-1 and in wild-type plants thereby ruling out an essential involvement of SOP2 and SOP2h in plant defence (Fig. 12B,C).  4.3.5 Dosage-dependent suppression of prl1-2 by a loss-of-function mutation in SOP2 supports dominant-negative activity of sop2-1D Since the dominant sop2-1d allele suppresses prl1 phenotypes, we were interested in finding out whether use of the loss-of-function allele (sop2-2) would restore wild-type phenotypes as well. Phenotypic changes were already observed in the F1 generation of the cross, among the plants with homozygous prl1-2 - and heterozygous sop2-2/SOP2 configuration. A representative plants produced segregation ratios of 45:19:22 (intermediate:WT-like:prl1-like), suggesting 67  semi-dominance of the sop2-2 allele (expected 2:1:1, χ2 = 0.4, P = 0.82). PCR-based genotyping identified homozygous prl1-2 sop2-2 mutants among the plants with wild-type traits and intermediate specimen as plants with heterozygous sop2-2/SOP2 configuration (Fig. 13A). The observed genetic ratios and phenotypes indicate dosage-dependent suppression of prl1-2 by loss-of-function of SOP2.  To test whether a homozygous sop2-2 genotype is functionally equivalent to sop2-1D activity, we compared resistance levels displayed by prl1-2 sop2-1D and prl1-2 sop2-2 double mutants three days after inoculation with the established virulent inoculum. As shown in Fig. 13B, lossof-function of SOP2 restored wild-type levels of resistance in prl1-2 sop2-2 double mutants, comparative to the defence output displayed by prl1-2 sop2-1D plants. This trend is consistent with morphological data showing similar root lengths for wild-type, prl1-2 sop2-1D and prl1-2 sop2-2 double mutants plants (Fig. 13C). In contrast, mutating the SOP2 homolog did not affect morphology nor defence and this could indicate functional diversification of SOP2h-1(Fig. 13B,D).  Taken together, our findings are consistent with semi-dominant suppression of prl1 mutant phenotypes by sop2-2, which further strengthens our hypothesis of a dosage dependent dominant-negative mechanism for sop2-1D.  4.3.6 SOP2 gene expression is only slightly altered in MAC mutants Public databases rank SOP2 expression at 25% and SOP2h at 15% of average deposited gene profiles. In contrast, MAC genes PRL1, MOS4, AtCDC5, MAC3a/MAC3b and MAC5a are highly expressed, at levels ranging from 1.4 to 2.8 times the average gene expression (Ace View: www.ncbi.nlm.nih.gov/IEB/Research/Acembly). To address a potential role of PRL1 or the MAC in regulating SOP2 gene expression we examined SOP2 mRNA levels in prl1-2 as well as in 68  mos4-1, atcdc5-2, mac3a mac3b and mac5a plants. Quantitative RT-PCR revealed unchanged levels of transcript in mos4-1 and mac5a mutants whereas slight elevated expression was detected in atcdc5-2 and mac3a mac3b mutants. In contrast, SOP2 expression in prl1-2 mutants is reduced, however not below 35% of wild-type levels (Fig. 13E). Suppression data thus do not support substantial regulatory control of MAC member over SOP2 gene expression although a minor role of PRL1 and potentially AtCDC5 might be possible.  4.3.7 SOP2 is a nuclear protein Since PRL1 and other members of the MAC localize to the nuclear compartment, a corresponding localization for SOP2 is a reasonable hypothesis (Palma et al., 2007; Nemeth et al., 1998). Recently, SOP2 was found in a large-scale analysis of chloroplast preparations, however current prediction programmes do not detect a reliable sorting signal for this compartment (Zybailov et al., 2008). Using an algorithm which specializes on importin αdependent nuclear import however, a putative nuclear localisation signal (NLS) was calculated for positions 50 to 79 of the SOP2 amino acid sequence, supporting co-localisation with MAC members (Fig. 10) (Kosugi et al., 2009). To visualize the intracellular distribution of SOP2, we created a C-terminal SOP2-GFP fusion construct encompassing 1kb of 5’ regulatory sequence and the SOP2 open reading frame. Transformation of the construct into prl1-2 sop2-1D resulted in six T1 transgenics, four of which exhibited prl1-like or intermediate phenotypes (Fig. 14A). Complementing, BASTA® resistant T2 progeny of prl1-like GFP-lines exhibited susceptibility similar to prl1 single mutants in infection experiments demonstrating, that the SOP2-GFP fusion protein functions properly and should be localized to its normal subcellular compartment (Fig. 14B). Although observed SOP2-GFP fluorescence was very weak, the fusion protein was detected in the nucleus using confocal microscopy (Fig. 14C). Low intensity of the GFP signal is probably due to low expression levels mediated by the genomic SOP2 promoter.  69  4.3.8 sop2-2 does not suppress MAC mutations atcdc5-2 or mos4-1 To test whether mutations in SOP2 affect signalling mediated by other MAC components, we examined double mutants of sop2 crossed with loss-of-function alleles of transcription factorencoding CELL DIVISION CYCLE 5 (AtCDC5) and predicted structural member MOS4 (Palma et al., 2007). Since both sop2-1D and sop2-2 were able to suppress prl1 phenotypes, we decided to use the insertion allele sop2-2 which allowed efficient PCR-based genotyping of atcdc5-2 sop2-2 and mos4-1 sop2-2 double mutants. If SOP2 functions in a MAC dependent pathway, the double mutants should resemble a wild-type plant analogue to the suppression phenotypes observed in prl1-2 sop2-2 plants. However, atcdc5-2 sop2-2 double mutants resemble atcdc5-2 single mutants, indicating that ATCDC5 and SOP2 act independently (Fig. 15A). Similarly, mos4-1 associated phenotypes such as broad leaves and a late flowering phenotype are still evident in the mos4-1 sop2-2 double mutant (Fig. 15B). Considering that a mutation in SOP2 solely suppresses prl1 phenotypes and does not seem to impact signalling in atcdc5-2 sop2-2 and mos4-1 sop2-2 plants, a function of SOP2 specific to PRL1 appears likely.  4.4  Discussion  We identified the dominant sop2-1D mutant from a prl1 suppressor screen aimed to identify signalling components downstream of the MAC, a multi-protein complex with a role in plant immunity. We found that SOP2 functions in PRL1-dependent signal relay but independent of two other MAC genes, AtCDC5 and MOS4. This suggests that SOP2 is not a target of the MAC but rather specific to PRL1. The PRL1 protein may operate only temporarily as part of the MAC, a hypothesis that is consistent with research demonstrating PRL1 activity in sugar, hormonal and abiotic stress related pathways (Flores-Perez et al., 2010; Baruah et al., 2009; Abraham et al., 2003; Nemeth et al., 1998). If PRL1 functions as a flexible facilitator of multiple protein interaction, SOP2 may in fact be part of yet another complex. 70  Mutations in PRL1 result in compromised resistance, a phenotype that is no longer observed in plants that also carry defects in the SOP2 gene. In mechanistic terms, PRL1 acts as a positive regulator and is required for sound resistance; SOP2 on the other hand appears to contribute negatively to resistance. Increased resistance towards fungal pathogen Golovinomyces cichoracearum (powdery mildew) and upregulation of defence related genes has been established for edr1 (enhanced disease resistance 1,) a negative regulator of resistance in Arabidopsis. The gene encodes a protein kinase possibly targeting transcription factors to the proteasome (Christiansen et al., 2011; Frye et al., 2001). Transcription factors themselves also may act as negative regulators; loss of WRKY11 and WRKY17 increases resistance to virulent and avirulent P.s.t. strains (Journot-Catalino et al., 2006). However, neither dominant sop2-1D nor recessive sop2-2 mutants displayed any signs of compromised resistance (increased or decreased) in our infection assays, which leaves the nature of SOP2 contribution to resistance currently unsolved.  A possible explanation for a lack of phenotypes might be functional redundancy between SOP2 and other homologs. We included a mutant allele of the only homolog SOP2h-1 and the sop2-2 sop2h-1 double mutant in our analysis to address redundancy but again recorded defence outputs similar to wild-type plants. mac5a, a putative RNA binding protein and component of the MAC does not exhibit susceptibility in pathogen infections but partially suppresses snc1associated phenotypes suggesting a contributing function in plant immunity (Monaghan et al., 2010). A snc1 sop2-2 double is currently generated in our laboratory and should address involvement of SOP2 in the constitutive defence response triggered in this mutant background.  Expression levels of SOP2 in a wild-type plant are approximately 80% lower than those of PRL1. Further reduction of functional protein amounts revealed a dosage-dependent relationship between SOP2 and PRL1 in heterozygous mutant plants and identified the sop2-2 loss-of-function allele as semi-dominant. We have thus identified a haplo-insufficient gene 71  acting in PRL1 signalling, for which the product of both alleles is needed to confer normal operations (Mao et al., 2011; Wang et al., 2008; Pillitteri et al., 2007). Intriguingly, the phenotype is only visible in the prl1 mutant background. Semi-dominant as well as dominant mutations have often been found in resistance genes, i.e. NB-LRR type R-proteins SSI4, SNC1, SLH1 and CHS3 in Arabidopsis and NLS1 in rice (Tang et al., 2011; Yang et al., 2010; Noutoshi et al., 2005; Zhang et al., 2003; Shirano et al., 2002). In these cases, sequence modifications usually led to upregulation of signalling responses and increased resistance, none of which were observed in sop2-2. Additionally, as a genetic suppressor of prl1, the SOP2 gene likely functions further downstream in a signal cascade.  Since the insertional T-DNA allele sop2-2 suppressed prl1 phenotypes, we hypothesized that sop2-1D was in fact also a loss-of-function mutation, however dominant. We observed a range of intermediate phenotypes among transgenic pr1-2 sop2-1D expressing wild type SOP2 and in prl1-2 mutants expressing sop2-1D. These phenotypes suggest a sop2-1D-encoded protein causing a dosage-dependent dominant negative effect. Association of SOP2 with other proteins in form of an oligomer or complex is one hypothesis that could explain the displayed intermediate morphology in transgenics as well as dosage dependency of prl1 suppression. Full functionality of a putative multi-subunit conglomerate might rely on sufficient amounts of SOP2 and such an oligomer could be sensitive to a spoiler version of SOP2. Increased amounts of one complex component would not be expected to result in a mutant phenotype due to stoichiometric interactions and is consistent with an observed absence of phenotypes in SOP2 overexpressing lines. The observation, that SOP2 is expressed at very low levels in the plant yet able to suppress prl1 signalling completely when defect, may further support this hypothesis.  Nineteen proteins of the MAC share homology with proteins of the yeast NINETEEN COMPLEX (NTC) and human complex CELL DIVISION CYCLE 5-LIKE-SENESCENCE EVASION FACTOR (CDC5L) (Monaghan et al., 2009). In the latter two systems, complex members are 72  either components of or are associated with the spliceosome, a connection that likely extends to the plant complex (Johnson et al., 2011). We hypothesized that targets of the MAC would be found among similarly conserved proteins; however homologs of SOP2 appear to be plant line exclusive. Using the PLAZA platform we identified homologous sequences in crop plants such as rice, soybean, apple, corn and cacao but also in poplar and in the model legume plants Medicago truncatula, Lotus japonicas and the grass Brachypodium distachyon (Proost et al., 2009). Although PRL1 is conserved among eukaryotes, its binding partners need not necessarily fall into the same category. WD40 proteins are versatile in their interactions with other proteins (Stirnimann et al., 2010). The majority of amino acid sequence of both PRL1 and close homolog PRL2 are taken up by seven WD40 repeats which assemble into a flexible binding interface (Xu and Min, 2011). Both PRL1 and close homolog PRL2 likely interact with a range of proteins and seem to be a more likely signalling partner of SOP2 by themselves than the conserved complex as a whole (Weihmann et al. submitted).  We detected SOP2-GFP fluorescence in the nucleus in line with a proposed NLS signal situated among the first 51 N-terminal amino acids. However, with a molecular weight of 58kD, SOP2 falls within the diffusion limit of the nuclear pore complex and does not necessarily need an NLS (Nardozzi et al., 2010; Wang and Brattain, 2007). Localising to the same subcellular compartment, SOP2 and PRL1 could potentially interact directly. Although we did not confidently identify a known domain among the SOP2 and SOP2h protein sequences, a novel plant specific motif might have not been recognized.  PRL1 belongs to a subset of WD40 proteins that also contain DWD (DDB1 binding WD40) motifs. Named after the UV-DAMAGED DNA BINDING PROTEIN1, this domain is credited to facilitate binding between DDB1 and diverse receptor proteins, including PRL1, as part of the Arabidopsis CUL4 E3 ubiquitin ligase (DCX - type)(Zhang et al., 2008; He et al., 2006). It is tempting to speculate a role for SOP2 in the ubiquitin proteasome pathway. We did not find a 73  strong correlation between PRL1 protein and SOP2 transcription levels however association between SOP2 and PRL1 remains to be explored.  74  A  WT  prl1-2  B  C 40 2  8 log cfu/cm  30 20  **  0  Day 0  Day 3  ** 6 4 2  pr W l12 s prl T op 1-2 21D  W T p pr r l 1l12 2s op 21D  0 W so prl1-T p2 2 -1 D  10  P.s.m. ES4326  pr l12  Average root length [mm]  prl1-2 sop2-1D  Figure 8. prl1 phenotypes are suppressed in prl1-2 sop2-1D (A) Morphology of 5-week-old soil grown plants of WT (Col-0), prl1-2 and prl1-2 sop2-1D. (B) Root length analysis of 1-week-old seedlings of indicated phenotypes. The results represent an average of 10 seedlings each ±SD. (C )Infection of 5-week-old plants (OD600= 0.0001) of indicated phenotypes and quantification of titer at 0 and 3 days post inoculation. Values represent averages of six replicates ±SD. Statistical significance compared to Col-0 control was calculated using Student’s t-test: **P < 0.0001 for both graphs.  75  A  D  prl1-2 sop2-1D  E  prl1-2  SOP2g in prl1-2 sop2-1D  P.s.m. ES4326 Day 0  log cfu/cm2  8  Day 4 **  **  6 4 2  1-2  At2g40630/SOP2  prl  B  W prl T so 1 -2 p2 p rl S 1-2 O P 1D so 2g i p2 n -1 D W prl 1-2 pr T l so 1 -2 p2 p rl 1-2 S O P 1D 2 so p 2 g in -1D  0  sop2-1D  sop2-2  F  C F L S G L L G G V A sop2-1D TTTTCTATCAGGCCTTTTGGGTGGAGTTGC SOP2 TTTTCTATCAGGCCCTTTGGGTGGAGTTGC F L S G P L G G V A WT amino acids 218-227  prl1-2  sop2-1Dg in prl1-2  Figure 9. Map-based cloning of sop2-1D (A) Positional mapping locates sop2-1D on the bottom arm of chromosome 2. Indicated are recombinants, markers and BAC clones as well as their respective sequence-anchored positions. A mutation (*) was identified in At2g40630. (B) At2g40630 was named SUPPRESSOR OF PRL1, 2 (SOP2), the sop2-1D mutation is located in the third exon. The T-DNA insertion site in sop2-2 (Salk_058710(C) Sequence analysis reveals a C to T transition in prl1-2 sop2-1D resulting in an amino acid change from proline to leucine. (D) Morphology of prl1-2 sop2-1D, prl1-2 and transgenic prl1-2 sop2-1D mutants expressing the genomic wild-type SOP2 gene. (E) Bacterial infection of the indicated phenotypes with P.s.m. ES4326. Plants were inoculated with a low dose (OD600= 0.0001) and titer quantified at 0 and 4 days past inoculation. Values represent averages of six replicates ±SD, Infections were repeated four times with similar results. Statistical significance relative to the wild-type was calculated with a Students t-test: **P < 0.0001 (F) Morphology of Col-0, prl1-2 and transgenic prl1-2 mutants expressing the genomic sop2-1D mutant allele (T1).  76  bipartite NLS  bipartite NLS  coiled-coil domain  coiled-coil domain  Figure 10. SOP2 and SOP2h are homologous proteins Sequence alignment with identical amino acids shaded in black and similar amino acids indicated in light grey. Predicted nuclear localization signals (NLS) and coiled-coil domains are shown for SOP2 above and for SOP2h below the alignment (Kosugi et al., 2009; Rattei et al., 2010).  77  ZM1900 55  BD2960 HV2094 OSI3280  89 67  100  100  70 87  OSJ3940 ZM8640 ZM0960 SB2670 RC0140 VV9220  RC AL BD CP FV GM HV LJ MD ME MT OSI OSJ PT SB TC VV ZM  MD7900 100  100 90 87  87  BD2960 MD2880 FV1180 LJ3580 MT7460  69  GM6380 82  GM6540  ME0010 RC1130 ME0160  87 69 93 84  PT7500 PT9010 TC6250 CP0610  100  Ricinus communis, castor oil plant Arabidopsis lyrata Brachipodium distachion, model grass Carica papaya, papaya Fragaria vesca, woodland strawberry Glycine max, soybean Hordeum vulgare, barley Lotus japonicas, model legume plant Malus domestica, apple Manihot esculenta, cassava/manioc Medicago truncatula, model legume plant Oryza sativa ssp indica, rice Oryza sativa ssp japonica, rice Populus trichocarpa, poplar Sorghum bicolor, sorghum Theobroma cacao, South American cacao Vitis vinifera, grape vine Zea mays, corn  RC0130 96 0.7  SOP2h (A. thaliana) AL4810 100  SOP2 (A. thaliana) AL7540  Figure 11. Phylogenetic analysis of SOP2 homologs in plants A phylogenetic tree based on maximum likelihood analysis was constructed from 31 plant protein sequences. Numbers indicate bootstrap support values expressed as a percentage for 500 bootstrap replicates; bootstrap supports below 50% are not shown. The scale represents the rate of amino acid substitutions. The tree was rooted at midpoint. For details, see Materials and Methods  78  A  WT  sop2-2  sop2-1D  C  B WT  prl1-2  P.s.m ES4326  sop2-2  log cfu/cm  2  6  sop2h-1  Day 0  Day 0  *  4  2  C  sop2-2 sop2h-1  W p T so rl1-2 p2 so -1D so p2 so p2-2 p2 2 so h p2 1 h1  sop2h-1  W p T so rl1p2 2 so -1D so p2 so p2-2 p2 2 so h p2 1 h1  0  Figure 12. Mutations in SOP2 and SOP2h do not cause morphological or disease-related phenotypes (A) and (B) Morphology of 5-week-old soil-grown plants of indicated genotypes. (C) Infection of Col-0, prl1-2, sop2-1D, sop2-2, sop2h-1 and sop2-2 sop2h-1 mutants with virulent P.s.m. ES4326. Leaves were inoculated with a low dose (OD600= 0.0001) and bacterial titer quantified at 0 and 3 days post inoculation. Values represent averages of six replicates, statistical significance relative to the wild-type plant was calculated using Students t-test: *P < 0.002. Experiments were repeated at least three times with similar results.  79  A  B  P.s.m. ES4326 Day 0  8  Day 0  prl1-2 sop2-2  log cfu/cm  2  **  prl1-2 SOP2/sop2-2  6  **  4 2  prl1-2  D WT  prl1-2  E  30 20  A  10  **  2.5 unscaled expression  Average root length [mm]  40  pr W l1p T pr 2 so rl1l pr 1-2 p2-1 2 l1- so D 2 s p2 op -2 2h -1  C  pr W l1p T pr 2 so rl12 l pr 1-2 p2-1 l1- so D 2 s p2 op -2 2h -1  0  2.0 1.5 1.0 0.5 0  prl1-2 sop2-2  prl1-2 sop2h-1  W T pr l1mo 2 s4 atc -1 ma d c3 c5am 2 ac 3 ma b c5 a  W T pr pr l12 s l1-2 op 2pr 1D l12s op 22  0  2  Figure 13. sop2-2 is a semi-dominant suppressor of prl1-phenotypes (A) Morphology of five-week-old soil-grown plants of prl1-2 sop2-2, prl1-2 SOP2/sop2-2 and prl1-2. (B) A low dose (OD600= 0.0001) of P.s.m. ES4326 was infiltrated into rosette leaves of indicated phenotypes and bacterial titer quantified after 0 and 3 days past inoculation. Values represent an average of six replicates ±SD. Analysis was repeated at least three times. (C) Root length analysis of 1week-old seedlings of indicated phenotypes. The results represent an average of 10 seedlings each ±SD. Experiment was repeated twice. (D) Morphology of 5-week-old soil-grown plants of indicated phenotypes. prl1-2 sop2-2 is phenotypically wild-type whereas the prl1-2 sop2h-1 mutant resembles prl1-2. (E) Quantitative RT-PCR analysis of SOP2 expression in MAC mutants. Values are normalized to tubulin expression. Experiment was repeated twice with similar results. Statistical significance for (B) and (C) was calculated using a Students t-test: **P < 0.0001 for both graphs.  80  B  A  P.s.m. ES4326 Day 0  Day 3  8  **  6  **  4 2  C  SOP2-GFP in prl1-2 sop2-1D  GFP  PI  W pr T l12 s prl1 op -2 S 2 p r OP l1- 2- -1D 2 s GF op P i 2- n 1D  prl1-2 sop2-1D  W pr l1p T 2 s rl1op 2 S 2 p r OP l1- 2- -1D 2 s GF op P i 2- n 1D  0  Merged 2  20µm  20µm  20µm  Figure 14. SOP2 localizes to the nucleus 20µmmutant and transgenic prl1-2 prl2-1D plants expressing a genomic (A) Morphology of prl1-2 prl2-1D SOP2-GFP fusion protein (B) Bacterial infection of the indicated phenotypes with virulent P.s.m. ES4326. Statistical significance was calculated using a Students t-test: **P < 0.0004 (C) Fluorescence in guard cells of transgenic plants was observed using confocal microscopy, cell walls were stained using propidium iodine (PI).  81  B  A WT  atcdc5-2  sop2-2  sop2-2 atcdc5-2  -1  so p2 -  2  m  os 4  1 m os 4-  so p2 -2  W  T  atcdc5-2 sop2h-1 atcdc5-2  Figure 15. sop2-2 does not affect mutant phenotypes of atcdc5-2 or mos4-1 (A) Morphology of five-week-old soil- grown plants of indicated phenotypes. (B) Morphology of fully grown Col-0, sop2-2, mos4-1 and sop2-2 mos4-1 plants.  82  5  Concluding discussion and outlook  The main goal of this thesis is to deepen our understanding of signalling steps leading to plant resistance. In particular, signalling associated with PRL1, a member of the MAC, has been at the centre of the presented research.  5.1  Suppressors of prl1 signalling  We set out to genetically dissect PRL1 signal relay since we hypothesized a considerable contributing role of this protein to plant defence. To find new components of the desired pathway, we carried out two suppressor screens with the goal of introducing mutations in genes downstream of PRL1 and potential targets of the MAC. Chronologically, the EMS screen was executed first. We succeeded in producing both complete and a range of partial morphological suppressors, most of which displayed restored R protein mediated resistance when exposed to an established Arabidopsis pathogen thus demonstrating strong correlation between morphological and disease-related phenotypes.  During preliminary examination of the complete suppressors, we established dominant inheritance for two of these mutants. This finding prompted us to execute a second screen based on Agrobacterium mediated insertional T-DNA mutagenesis. The rationale for this decision lies in the accelerated cloning possibilities for dominant mutations caused by a loss-offunction mechanism. As introduced in Chapter 2, a portion of Arabidopsis genes fall into the haplo-insufficient category thus causing a dominant or semi-dominant phenotype with functional loss of already one allele copy (Mao et al., 2011; Wang et al., 2008; Pillitteri et al., 2007).  Haplo-insufficient genes in a pathway can also be identified through insertional mutagenesis followed by PCR-based protocols, referred to as T-DNA tagging. This cloning approach can 83  identify a mutated haplo-insufficient gene faster than conventional map-based cloning techniques. It is unfortunate that we were not able to obtain mutants through this screen. Closer examination of SOP2 revealed this gene to be indeed haplo-insufficient and thus it could have been identified through T-DNA tagging.  Optimized environmental conditions are vital to the success of a screen, and they were not ideal in our studies because soil-borne an air-borne pathogens infested our growth chambers. These circumstances visibly increased stress levels in prl1 plants, which are both stress sensitive and highly susceptible. Considering these observations, it becomes clear that the choice of mutagen is important when dealing with mutants that are less robust than a wild-type plant. Transformation efficiencies are affected by the health of the plant and thus chemical (EMS) mutagenesis, carried out during the seed stage, is recommended for future screens using sensitive mutants. Additionally, single nucleotide polymorphisms often produce a range of mutation, i.e., we created not only a dominant negative but also a dominant gain-of-function mutant and a collection of recessive mutants.  The scope of this thesis does not include comprehensive complementation analysis which leaves the number of loci among the remaining sop mutants undetermined. The SOP2 gene was sequenced in the three semi-dominant mutants sop15, sop17 and sop19 that we obtained, with the hope of finding additional alleles. However, the phenotypes in these mutants appear to be caused by mutations at different site(s). Further adding to the potential number of sop loci are the recessive mutants. Crosses among them should be undertaken to establish overall complementation group numbers for future mapping projects.  Although designed similar to the snc1 screen, less affected loci are expected among the obtained prl1 suppressors. The deregulated, predicted resistance protein in snc1 is positioned at the beginning of a defence network, constantly emitting danger cues (Zhu et al., 2010; Li et 84  al., 2001). PRL1, on the other hand, is a structural protein presumably functioning further downstream in the pathway and for which we used available recessive alleles in the screen. Since we are narrowing in on signalling steps further downstream in a signal cascade, the scope of defence components that are potentially involved also becomes understandably more limited.  5.2  Analysis of PRL2  The suppression caused by sop1-1D/prl2-1D is due to a dominant gain-of-function mutation in the homolog of PRL1. The mutation however is not located in the protein-coding open reading frame – as is the case for snc1 - but rather just upstream of the translational start codon. Consequentially, the encoded PRL2 protein is unaffected. A gain-of-function mutation may alter a gene’s function or expression patterns, in both cases diverging from the natural profile and resulting in a dominant phenotype. sop1-1D/prl2-1D is an example of a regulatory mutation, dramatically altering gene expression.  PRL2 had been previously identified as a close sequence homolog to PRL1 and therefore functional overlap of the encoded proteins is a reasonable assumption (Nemeth et al., 1998). However, in contrast to the pleiotropic effects displayed by prl1 mutants, the prl2 mutants that have been tested are indistinguishable from wild-type plants and thus have not allowed functional characterization of the homolog. Our attempts to prove that an allele of PRL2 is the suppressor in the prl1-2 sop1-1D double mutant have been laborious. For unknown reasons we were unable to create a PRL2 specific primer pair which would result in sufficient amounts of PCR product. By attempting to amplify only half of the PRL2 gene, we determined that the genomic region upstream of the open reading frame - and in which left primers were binding was problematic. It would be interesting to determine the cause of his problem. Is the chromatin  85  in this region organized tighter or differently in prl1-2 sop1-1D than in prl1-2 or a wild-type plant? Could there be steric hindrance of the utilized polymerases?  These questions tie in with the yet unknown mechanism by which gene expression in sop11D/prl2-1D is enhanced. Potentially, a cis-acting (promoter) motif influencing transcription rate has been altered. Such an element could be involved in recruiting transcription factors or other related proteins to the PRL2 gene. Enhanced binding of a factor or alternatively, reduced binding affinity for an inhibitor of transcription could be envisioned. Mutating a controlling motif may have substantial impacts on overall expression patterns.  Transcript levels could also be affected post-transcriptionally. If stability of a given mRNA molecule is increased, more protein product can be synthesized through repeated translation events. Fluorescence in situ hybridization or radioactive labelling of transcripts could be attempted to determine whether increased transcription or stability-enhanced RNA molecules are present in the prl2-1D mutant.  Using JAtY clone 69M23 and a transformation protocol that had been substantially modified during the T-DNA screen, we succeeded in creating transgenics that carried two PRL2 alleles in the prl1 homozygous background. Although only three transgenics were identified, the phenotypes supported our hypothesis and we proceeded with the comprehensive analysis of PRL2. The two WD40 proteins are functionally equivalent, suggesting that the less conserved Cterminal regions are not essential for protein interaction. As previously noted, sequences adjacent to the PRL2 open reading frame have however diversified significantly and are likely responsible for the observed unequal genetic redundancy. After a gene duplication event, expression patterns of one or both copies may be altered to compensate for undesired dose effects (Yang et al., 2011; Schuster-Bockler et al., 2010; Zou et al., 2009). We did record 86  enhanced phenotypes in prl1-2 prl2-2 double loss-of-function mutants, confirming at least residual activity of PRL2 in a wild-type plant. Consequently, PRL1 expression might be lower now than before the assumed duplication event, to accommodate for remaining PRL2 activity. A dynamic relationship would also leave room for the development of specialized patterns, such as a suggested tissue specificity for PRL2.  Most likely due to low abundance, PRL2 has not been detected in the recent immunoprecipitation of the MAC (Monaghan et al., 2009). Nonetheless, PRL2 is probably regularly incorporated into a small number of MAC complexes and acting interchangeable with PRL1 in resistance. Other phenotypes typical for prl1 mutants, i.e. sugar sensitivity and stunted roots are also suppressed by prl2-1D which serves as evidence towards equivalence beyond defence signalling. PRL2 likely acts as an equally capable regulator of sugar, hormone and stress influenced genes, functions which have been repeatedly associated with PRL1 (FloresPerez et al., 2010; Baruah et al., 2009; Li et al., 2007; Bhalerao et al., 1999; Nemeth et al., 1998; Salchert et al., 1998).  5.3  Discovery of SOP2  sop2-1D was pinpointed using a positional cloning approach and followed by candidate sequencing analogue to mapping of prl2-1D. A large number of conserved genes with predicted homologs across kingdoms were found among the twenty-five candidates present in the flanked region. We chose to sequence these genes first, in line with the evolutionary conservation of the MAC, however we did not find a sequence polymorphism. Thereafter we investigated genes with predicted functions conceivable as targets of PRL1 or the MAC. Eventually, the last two genes which were positioned closest to one of the flanking makers were analysed, and the sop2-1D mutation was found in a plant specific gene with unknown function. This finding was  87  unexpected and underlines the need for constant reassessment of hypotheses in our efforts to unravel pathways.  SOP2 does not fit our profile of a MAC target. Epistasis analysis later revealed that SOP2 functions in PRL1 signalling but is not part of AtCDC5 or MOS4 signal transduction, two other core MAC components. In this context, SOP2 as a target of PRL1 only is a more likely hypothesis considering the plant specific nature of the suppresser gene and the high level of conservation for the MAC as a complex. A versatile adaptor protein may facilitate interaction and interact with a range of partners, independent of their level of overall conservation. PRL1 is largely comprised of WD40 repeats, a prominent motif in plants (Stirnimann et al., 2010).  All investigated members of the MAC are expressed at levels well above average gene data in the AceView database, PRL1 ranks at 140% of average levels. In contrast, SOP2 and even more so, SOP2h, are expressed at very low levels. This added a complication to efforts of localizing a SOP2-GFP fusion protein and left us with the need to explain how mutations in SOP2 are able to suppress prl1 signalling so completely. Could a rate limiting step be involved in which a small number of SOP2 molecules control subsequent signalling? Are SOP2 and PRL1 associated as an oligomer? Or is this dependency not based on mutual protein activity? We tested transcription levels of SOP2 in all core MAC mutants but did not find strong evidence of a regulatory relationship existing on the transcription level. In a next step, we have initiated a cross between transgenic prl1-2 sop2-1D plants expressing genomic SOP2-GFP and a double mutant of MAC3a and MAC3b, both MAC core components with predicted E3 ubiquitin ligases activity. If SOP2 is post-translationally modified by MAC3a/3b, absence of ubiquitination could lead to higher observable GFP fluorescence.  Despite our efforts, SOP2 function remains elusive. We ordered T-DNA mutants to examine whether loss-of-function would result in a mutant phenotype however single and double mutants 88  of SOP2 and homolog SOP2h are largely wild-type in appearance. Additionally, none of the mutants exhibited increased resistance or related defects when exposed to pathogens. These findings argue against a possible role of the homologs as negative regulators. It should be established next, whether SOP2 functions directly in immunity signalling. All investigated members of the MAC, except for the untested relationship with PRL1, suppress snc1 signalling fully or partially. SNC1 and PRL1 are located in close proximity on chromosome 4 and because of this genetic linkage, a double mutant could not be created for use in such an analysis (Palma et al., 2007). Thus, examination of a snc1 sop2-2 double mutant would address several questions: a) if wild-type morphology is at least partially restored, SOP2 would be considered a component of snc1 resistance signalling with a potential role in overall immunity and b) if this is the case, a so far theorized ability of prl1 to suppress the constitutive resistance signalling in snc1 would be assured.  Endorsement of SOP2 as a defence molecule should lead to further investigation, i.e. crossing the mutant with SA-deficient mutants and npr1, a major regulator in resistance. To clarify the role of SOP2 relative to SA - dependent signalling, measurement of SA levels in sop2 mutants could be a first step. Also, crossing of sop2 with sid2 or eds5 mutants could be attempted and resistance levels of double mutants analysed. To evaluate the relationship between SOP2 and NPR1, sop2 npr1 or sop2 npr1 snc1 mutants could be generated and levels of marker genes observed, i.e. PR2/BGL2 expression is a hallmark of the NPR1-independent pathway.  Two lines of evidence support a dominant negative mutation in sop2-1D. Classical examples for this type of mutation have been factors that oligomerize or associate as homodimers and in which the mutant form acts as a spoiler protein. Disruption of structural polymers is another example (Veitia, 2007). SOP2 however is also a haplo-insufficient gene, a phenotype that has often been found in enzyme-encoding genes and transcription factors (Qian and Zhang, 2008; Seidman and Seidman, 2002). In an effort to learn more about the SOP2-encoded protein, a 89  yeast-two-hybrid or co-immunoprecipitation approach could be employed to identify potential binding partners. If SOP2 associates with other proteins, identities of such partners could serve as valuable clues towards SOP2 function.  PRL1 has been suggested to function as a substrate receptor, a part of the ubiquitin proteasome pathway in plants (Lee et al., 2008; Farras et al., 2001). Two DWD boxes, which are found embedded in the WD40 repeats, may be the motifs mediating interaction between PRL1 and DDB1 (DAMAGED DNA BINDING 1), a substrate adaptor for CULLEN 4-type (DCX) E3 ubiquitin ligases (Biedermann and Hellmann, 2011; Lee et al., 2008; Lee and Zhou, 2007). E3 ligases are multi protein complexes and potentially susceptible to a spoiler subunit, i.e. a dominant negative form of SOP2. However, defects in genes of DCX complex members, CULLEN 4, DDB1 and RBX1 lead to mutant phenotypes which we did not observe in sop2-1D or sop2-2 or in double mutants of SOP2 and homolog SOP2h (Lee et al., 2008; Lechner et al., 2002; Schroeder et al., 2002). Aside from being theoretically possible, participation of SOP2 in this important pathway is thus not supported from our data.  In conclusion, further investigation of SOP2 will deepen our understanding of PRL1 associated signalling in defence, dependent or independent of the MAC. Using the remaining sop mutants as a resource, a more complete picture of interactions facilitated by this WD40 protein can be revealed and might also lead to discoveries in signalling branches other than immunity.  90  References Aarts, N., Metz, M., Holub, E., Staskawicz, B. J., Daniels, M. J. and Parker, J. E. 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To reduce these phenotypes and increase transformation efficiency, several modification were introduced (highlighted with * and in bold).   Grow 4 ml of O/N Agro culture 28-30ºC    Innoculate 300 mls of LB (+ appropriate antibiotics) with 50µl of O/N Agro culture, let grow to OD600 0.8-1.0 at 28-30ºC  1. Spin down 300 mls of culture at approximately 5,000 rpm in the Sorvall RC5C plus centrifuge (Rotor SLA-1500) for 15 minutes at RT 2. Resuspend in 300 mls of 5% sucrose and 0.01% wetting agent (Silwet) 3. Spray plants *only briefly to minimize weight on aerial parts 4. *Place plants in a darker area for 2-3 days, without direct light from the top. Do not cover plants with plastic bag. 5. *Transfer back to growth chamber but leave rack lights off for 5-8 days (Indirect light from neighbouring racks will trigger slower growth but less aborted seeds with desired mutations) The sucrose is made up fresh just before use (not sterile).  112  Appendix 2: Molecular marker InDel and SNP marker sequences used in the mapping of sop1 and sop2  Marker K14A17-F  Chr. Location 3  5.84Mb  K14A17-R MGL6-F  3  5.63Mb  MGL6-R MGL6-SNP1-Ler MGL6-SNP1-Col  3  5.66Mb  MGL6-SNP1-R MGL6-SNP5-Ler MGL6-SNP5-Col  3  5.69Mb  MGL6-SNP5-R MDC8-F  3  5.58Mb  MDC8-R MIE1-A  3  4.87Mb  MIE1-B MRC8-F  3  6.21Mb  MRC8-R T2N18-F  Col/Ler: T/C SNP (PERL0458534) 92bp fragment for both  5'-CTGGTACATTCACTTCCTTC-'3  Col/Ler: G/A SNP (PERL0458860) 173bp fragment for both  5'-AGAATGATGGTGGAGCTGAT-'3  Col: 557bp/Ler: 461bp 96bp InDel  5'-TGGCAAACTTGTTGGGTTCC-'3  Col: 450bp/Ler: 318bp 132bp InDel  5'-CTAAGTTCTTCCACCATCTG-3'  Col: 232bp/Ler: 246bp 14bp InDel  5'-GATGTCGGAATTGTCGATCG-'3  5'-CCCTTCATAACTAATTCCACACA-'3  5'-CTGGTACATTCACTTCCTTT-'3 5'-GAATTCAATGCCTCCGGTTA-'3  5'-AGAATGATGGTGGAGCTGTC-'3 5'-AACCTAATGCGGTCTACTGA-'3  5'-CATATGTGCCTTCAACTGCAG-'3  5'-CAAGGAGCATCTAGCCAGAG-'3  5'-TCGCAGAAACCACACTAAACC-3'  16.94Mb  Col: 95bp / Ler: 110bp 14bp InDel  5'-CGTACGTGAGAGATATGCAA-'3  Col: 469bp / Ler: 538bp 68bp InDel  5'-TTTCTTGGAAATTCGGGTTG-'3  Col: 443bp / Ler: 355bp 87bp InDel  5'-TGGTGTTGACGAACTTCCAA-'3  Col: 249bp / Ler: 231bp 17bp InDel  5'-GGCGTTTTTAATGGCAGTTC-'3  Col/Ler: C/G SNP (PERL0398101) 270bp fragment for both  5'-CGTTCTTTGTCTCTCTCTTAC-'3  2  16.86Mb  2  17.10Mb  2  17.01Mb  T7D17-SNP2-Col T7D17-SNP2-R  5'-CTTCAGCGGCTTGCATCTAT-'3  2  T7D17-R T7D17-SNP2-Ler  Col: 330bp / Ler: 384bp 53bp InDel  5'-CTTTGTTTGAAGTCGCATCG-'3  5’-TTGGTCACTAGTAAGATCTTG-3’  T3K9-R T7D17-F  5'-CCAAGCCTCTGCGTCTCTAC-'3  Col: 299bp/Ler: 227bp 72bp InDel  T3G21-R T3K9-F  Col: 397bp / Ler: 367bp 29bp InDel  15.57Mb  T2P4(2)-R T3G21-F  Sequence  2  T2N18-R T2P4(2)-F  Polymorphism:  2  17.03Mb  5’-GTCGTCTAGTGTACTTGTAGC-3’  5'-ATCACCAGATGGAAGTCTTG-'3  5'-AGTTTGAAGCCAAGCAAACG-3'  5'-TCGGAAGGAGCATTATGGAC-'3  5'-GCATTTACGGAAGCAGAAGG-'3  5'-CGTTCTTTGTCTCTCTCTTAG-'3 5'-AATGTGACCAAGACAACTTCC-'3  113  Appendix 3: Gene expression profiles of PRL1 and PRL2  Comparison of PRL1 (At4g15900) and PRL2 (At3g16650) expression patterns using the AtGenExpress visualisation tool. Data are derived from Affymetrics (Santa Clara, CA, USA) gene chip oligonucleotide arrays and are expression estimates by gcRMA. 114  Appendix 4: Gene ID’s of SOP2 homologs  Abbreviated ID  Full gene ID  Species  AL4810 AL7540 AT0630 AT5240 BD2960 CP0610 FV1180 GM6380 GM6540 HV2094 LJ3580 MD2880 MD3850 MD7900 ME0010 ME0160 MT7460 OSI3280 OSJ3940 PT7500 PT9010 RC0130 RC0140 RC1130 SB2670 TC6250 VV9220 ZM0960 ZM1900 ZM8640 ZM9510  AL6G04810 AL4G27540 AT2G40630 AT5G05240 BD3G02960 CP00019G00610 FV7G31180 GM14G26380 GM17G26540 HV326532094 LJ1G003580 MD04G002880 MD00G333850 MD00G137900 ME10895G00010 ME08315G00160 MT1G007460 OSINDICA_02G03280 OS02G03940 PT19G07500 PT13G09010 RC29976G00130 RC29976G00140 RC29729G01130 SB04G002670 TC10G016250 VV13G09220 ZM04G40960 ZM05G21900 ZM05G18640 ZM05G29510  Arabidopsis lyrata Arabidopsis lyrata Arabidopsis thaliana Arabidopsis thaliana Brachypodium distachyon Carica papaya Fragaria vesca Glycine max Glycine max Hordeum vulgare Lotus japonicas Malus domestica Malus domestica Malus domestica Manihot esculenta Manihot esculenta Medicago truncatula Oryza sative spp. indica Oryza sative spp. japonica Populus trichocarpa Populus trichocarpa Ricinus communis Ricinus communis Ricinus communis Sorghum bicolor Theobroma cacao Vitis vinifera Zea mays zea mays Zea mays Zea mays  115  Appendix 5: Gene expression profile of SOP2  Visualisation of SOP2 (At2g40630) expression patterns as available from AtGenExpress. Data are derived from Affymetrics (Santa Clara, CA, USA) gene chip oligonucleotide arrays and are expression estimates by gcRMA. SOP2h (At5g05240) is not present on the arrays. 116  

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