Institut für Nutzpflanzenwissenschaften und Ressourcenschutz (INRES) Lehrstuhl für Molekulare Phytomedizin Identification and characterization of a cystatin-like effector protein from beet cyst nematode Heterodera schachtii and its role in plant-nematode interaction Inaugural Dissertation zur Erlangung des Grades Doktor der Agrarwissenschaften (Dr. agr.) der Landwirtschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Marion Hütten aus Wesel, Deutschland Bonn, 2018 Angefertigt mit Genehmigung der Landwirtschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn. 1. Gutachter: Prof. Dr. Florian M.W. Grundler 2. Gutachter: Prof. Dr. Frank Hochholdinger Tag der mündlichen Prüfung: 04.12.2017 ii Table of Content I. Abbreviations vii II. Figures ix III. Tables x 1. Chapter 1 General introduction 1.1 Nematoda 1 1.1.1 Plant-parasitic nematodes 2 1.1.2 Cyst nematodes 5 1.1.2.1 Heteroderaschachtii 7 1.1.2.2 Life cycle 7 1.1.2.3 Management 10 1.2 Plant-nematode interaction 13 1.2.1 Morphological changes and molecular background of host cells during syncytium development 14 1.2.2 The role of effector proteins in plant-nematode interaction 16 1.3 Cysteine Proteases 18 1.3.1 Papain-like Cysteine Proteases (PLCPs) 19 1.3.2 Cystatins 20 1.4 Objectives 21 1.5 References 23 2. Chapter 2 Activity profiling reveals changes in the diversity and activity of proteins in Arabidopsis roots in response to nematode infection 2.1 Abstract 2.2 Introduction 2.3 Material and Methods 2.3.1 Plant and nematode culture 2.3.2 Activity-based protein profiling 2.3.3 Quantitative real-time PCR 2.3.4 Nematode infection assay 2.4 Results iii 2.4.1 Vacuolar processing enzymes (VPEs) 2.4.2 Serine hydrolases (SHs) 2.5 Discussion 2.5.1 Activities of vacuolar processing enzymes are reduced in the syncytium 2.5.2 Selective activation of serine hydrolases in the syncytium 2.6 References 3. Chapter 3 Papain-like cysteine proteases and proteasomal activities are suppressed in syncytium induced by cyst nematodes 3.1 Abstract 46 3.2 Introduction 46 3.3 Material and Methods 49 3.4 Results 50 3.4.1 Activity of papain-like proteases (PLCPs) is suppressed in female associated syncytium 50 3.4.2 Activity of proteasomal subunits is suppressed in female associated syncytium but not in male associated syncytium 51 3.5 Discussion 52 3.5.1 Papain-like cysteine proteases are inactivated in syncytium 52 3.5.2 Inhibition of proteasome in syncytium 53 3.6 Conclusion 54 3.7 References 59 4. Chapter 4 A cystatin-like effector protein of beet cyst nematode Heteroderaschachtiiperforms diverse roles during plant-nematode interaction 4.1 Abstract 67 4.2 Introduction 67 4.3 Material and Methods 70 4.3.1 Bioinformatic approaches 70 4.3.2 Expression of HsCysL1 in H. schachtii 70 4.3.3 Localization of HsCysL1 71 4.3.4 Y2H 72 4.3.5 BiFC 72 iv 4.3.6 Functional characterization of target genes 73 4.4 Results 73 4.4.1 Sugar beet cyst nematodes encode a cystatin-like protein 73 4.4.2 HsCysL1 is secreted into the host tissues 74 4.4.3 HsCysL1 shows diverse localization in plant 75 4.4.4 HsCysL1 interacts with PTPLA and UBC19 76 4.4.5 Knock-out of PTPLA does not affect nematode development 77 4.5 Discussion 77 4.6 Conclusion 82 4.7 References 91 5. Chapter 5 General discussion 5.1 Activity of vacuolar processing enzymes (VPEs) is reduced upon nematode infection 102 5.2 Serine hydrolases (SHs) are involved in metabolic processes during nematode infection 102 5.3 The plant proteasome constitutes a defence mechanism that gets circumvented by H. schachtii 104 5.4 Heterodera schachtii suppresses several papain-like cysteine proteases (PLCPs) to enable infection 105 5.5 Cystatins inhibit PLCPs 107 5.6 A putative effector found in H. schachtii obtains cystatin characteristics 107 5.7 HsCysL1 may has surprisingly dual function during syncytium establishment 108 5.8 Target genes of HsCysL1 reveal an involvement in signalling and regulatory processes 109 5.9 References 112 6. Summary xi 7. Zusammenfassung xii 8. Eidesstattliche Erklärung xiii 9. Danksagung xiv v vi ER – Endoplasmatic reticulum I. Abbreviations ETI – Effector-triggered immunity FLNs – Free living nematodes A. thaliana – Arabidopsis thaliana G - Glycine A. tumefaciens – Agrobacterium G. spp. – Globodera spp. tumefaciens GFP – green fluorescent protein ABPP – Activity-based protein profiling GOI – gene of interest ABPs – Activity-based probes H. spp. – Heterodera spp. AD – Activation domain H+-ATPases – hydrogen proton APNs – Animal parasitic Nematodes conjugating adenosine triphosphatases BD – Binding domain HR – Hypersensitive response BiFC – Bimolecular Fluorescence HsCysL1 – Heterodera schachtii Complementation Cystatin-like protein 1 BSA – Bovine serum albumin ISC – Initial syncytial cell CaMV – Cauliflower Mosaic Virus J2,3,4 – juvenile stages CathB – Cathepsin B kDa – protein molecular weight [kilodalton] CBB – Coomassie Brilliant Blue LAX3 – auxin influx transporter cDNA – Complementary DNA LRR – Leucine-rich repeats Cf– Cladosporium fulvum M. spp. – Meloidogyne spp. CN – cyst nematode MAPK – mitogen-activated protein CP – core protease kinase CRISP – cysteine-rich secretory MES – Methylesterase protein superfamily mRNA – messenger RNA DIG – Digoxygenin MS – Mass Spectrometry DMSO – Dimethyl sulfoxide N. benthamiana – Nicotiana DNA – Deoxyribonucleic acid benthamiana dNTPs – Deoxynucleotide NLS – nuclear localization sequence triphosphates NPC – Non-probe control dpi – Days post infection ON – Over night DTT – Dithiothreitol P. infestans – Phytophthora infestans EPIC – Extracellular protease inhibitor with cystatin-like domain P. syringae – Pseudomonas syringae vii PAMP – Pathogen-associated SFGH – S-formyl-glutathione molecular pattern hydrolase PBA1, PBB1, PBE1 – Catalytic SHs – serine hydrolases proteasomal subunits SP – signal peptide PCD – Programmed cell death TBS – TRIS-buffered saline PCR – Polymerase Chain Reaction T-DNA – Transfer-DNA PLCPs – Papain-like cysteine proteases TPP2 – Tripeptidyl peptidase-2 PPN – Plant-parasitic nematodes TRIS – 2-amino-2-(hydroxymethyl)-1,3- propanediol PR – Pathogenesis-related protein UBC19 – Ubiquitin-conjugating PTI – PAMP-triggered immunity enzyme 19 PTPLA – Protein-tyrosine V - Valine phosphatase-like A VAPs – Venome-allergen like proteins PVDF – Polyvinylidenfluoride VPEs – Vacuolar processing enzymes Q - Glutamine VS – Vinyl sulfone qRT-PCR – Quantitative real-time PCR W – Tryptophane qvpe – Quadruple VPE knockout line of A. thaliana WT – Wildtype RhFP – Rhodamine-tagged Y2H – Yeast-2-Hybrid fluorophosphonate probe YFP – Yellow Fluorescence Protein RNA – Ribonucleic acid YVAD-cmk – TYR-VAL-ALA-ASP- RNAi – RNA interference chloromethylketone - -1 RP – regulatory particle μEm ²s –microeinstein per square meter per second rRNA – ribosomal RNA S. cerevisiae – Saccharomyces cerevisiae SDS – sodium dodecyl sulfate viii II. Figures Chapter 1: Figure 1: Life cycle of H. schachtii 8 Chapter 2: Figure 1: Scheme of root sampling for ABPP analysis Figure 2: AMS101 labelling of the syncytium, root and leaf Figure 3: RhFP labelling of the syncytium, root and leaf Chapter 3: Figure 1: MV151 labelling of female associated syncytium, root and leaf 55 Figure 2: MV151 labelling of male associated syncytium and root 56 Figure 3: FH11 labelling of female associated syncytium and root 56 Chapter 4: Figure 1: Phylogenetic tree of HsCysL1-like homologues and sequence alignment 84 Figure 2:In situ hybridization and qRT-PCR showing the localization and expression of HsCysL1 85 Figure 3: Localization of HsCysL1 in plant 86 Figure 4: Y2H – Interaction of HsCysL1 with three target genes of Arabidopsis thaliana 87 Figure 5: BiFC – Confirmation of interaction of HsCysL1 with three target genes of Arabidopsis thaliana 88 Figure 6: Genotypic and functional characterization of PTPLA single knock-out line of Arabidopsis thaliana 89 ix III. Tables Chapter 2: Table 1: Gene expression of the VPEs in 5 and 15dpi syncytia. Chapter 3: Table 1: Quantitative RT-PCR of CathB transcription in 5and 10 dpi syncytia 57 Supplementary Table ST1: Affimetrix chip analysis data: PLCP transcription in syncytia (5 and 15 dpi) 57 Supplementary Table ST2: Affimetrix chip analysis data: Proteasome transcription in syncytia (5 and 15 dpi) 57 Chapter 4: Table 1: Putative interacting targets of HsCysL1 identified through Yeast-2-Hybrid screening 90 Supplementary Table ST1: Primer sequences used during experiments conducted in presented work 90 x Chapter 1 – General introduction 1. Chapter 1 General introduction 1.1 Nematoda Nematodes, unsegmented threadlike animals (Greek: nema = thread) belonging to the phylum Nematoda or Nemata, constitute one of the most diverse and abundant animal groups, inhabiting almost every ecological and parasitic niche. Some studies suggest that every four out of five animals on earth are nematodes [1]. Although the existence of animal and human pathogenic nematodes has been known for many centuries, plant-parasitic nematodes in particular long escaped discovery owing to their miniscule size 0.25 to 2 mm, which is impossible to detect without microscopic equipment. Indeed, when Turbevil Needham reported the first plant-parasitic nematode to the British Royal Society in 1743[2], he failed to gain the attention. In 1918, Nathan Augustus Cobb, a pioneer in the field coined the word “nematology” for this discipline [3], naming a new era for understanding the effects of nematodes on agricultural production. Yet nematodes were overlooked and underestimated for their impact for