AIX-MARSEILLE UNIVERSITÉ Ecole Doctorale Sciences de la Vie et de la Santé THÈSE DE DOCTORAT Spécialité: Génétique Presenté par: Le HE Pour obtenir le grade de docteur de l’Université Aix-Marseille Interactions hôte-pathogène entre Caenorhabditis elegans et le champignon Drechmeria coniospora Soutenue le 2 décembre 2016 devant le jury composé de: Dr. Dominique Ferrandon Rapporteur Dr. Hinrich Schulenburg Rapporteur Dr. Philippe Naquet Président Dr. Eric Record Invité Dr. Jonathan Ewbank Directeur de thèse I TABLE OF CONTENTS Table of Figures ................................................................................................................ IV Table of Tables .................................................................................................................. V CHAPTER 1. Introduction............................................................................................ 1 1.1 Host-pathogen interactions ................................................................................... 1 1.1.1 C. elegans and its innate immunity ............................................................... 1 1.1.2 Nematophagous fungus ................................................................................. 5 1.1.3 Plant pathogenic fungi ................................................................................ 10 1.2 Fungal genetic modification ............................................................................... 17 1.2.1 Small RNA for Cross-species gene silencing ............................................. 17 1.2.2 Transformation ............................................................................................ 23 1.3 A D. coniospora effector and its possible host target ........................................ 29 1.3.1 Fungal effectors and saposins ..................................................................... 29 1.3.2 Ascorbate peroxidase fingerprinting-based effector discovery ................... 34 CHAPTER 2. Fungal genetic modification system .................................................... 36 2.1 Plasmid construction based on Gibson assembly and ccdB selection ................ 37 2.2 Article 1 .............................................................................................................. 39 2.2.1 Additional information: transgenic strains list ............................................ 46 2.3 Discussion and perspective ................................................................................ 47 II CHAPTER 3. Cross species RNAi in D. coniospora ................................................. 50 3.1 Primary small RNA for cross-species RNAi ...................................................... 51 3.2 Secondary small RNA for cross-species RNAi .................................................. 54 3.3 Discussion and perspective ................................................................................ 54 CHAPTER 4. D. coniospora effectors and its genomic sequences ............................ 57 4.1 Article 2 .............................................................................................................. 57 4.2 Additional information I- SapA-SapB protein interaction ............................... 108 4.3 Additional information II- Comparison between ATCC 96282 and ARSEF 6962 111 4.4 Using APEX to identify fungal effectors ......................................................... 113 4.4.1 Discussion and perspective ....................................................................... 119 CHAPTER 5. Materials and Methods ...................................................................... 125 5.1 Plasmid construction based on Gibson assembly and ccdB selection .............. 125 5.2 Fungal genetic modification system ................................................................. 126 5.3 SapA-SapB protein interaction ......................................................................... 126 5.4 Fungal effector identification from APEX worm strains ................................. 127 CHAPTER 6. Conclusion and perspective ............................................................... 115 6.1 Conclusion ........................................................................................................ 115 6.2 Perspective ....................................................................................................... 116 III CHAPTER 7. References.......................................................................................... 117 TABLE OF FIGURES Figure 1.1 Saprophytic and parasitic stages of the nematode-trapping fungus Arthrobotrys oligospora. .................................................................................................... 6 Figure 1.2 Tritrophic lifestyles of the nematophagous fungus P. chlamydosporia ........... 7 Figure 1.3 Parasitic lifecycle of D. coniospora .................................................................. 9 Figure 1.4 Diagram of pathogenic lifestyles.. .................................................................. 11 Figure 1.5 Graphical representation of standard internal operon processing ................... 21 Figure 1.6 One-step isothermal in vitro recombination with Gibson Assembly ............... 25 Figure 1.7 Schematic representation of the gene deletion ................................................ 28 Figure 1.8 Gene-for-gene interactions specify plant disease resistance. ........................... 30 Figure 1.9 Schematic diagram of prosaposin and SapA cleavage. ................................... 32 Figure 2.1 Reporter plasmid construction based on pLH4254.. ....................................... 36 Figure 2.2 Knock-out plasmid construction pLH4252.. ................................................... 38 Figure 3.1 RNAi feeding on rde-1 strain.. ....................................................................... 50 Figure 3.2 RNAi feeding on N2 and IG274 strain.. ......................................................... 52 Figure 3.3 Phenotype of strain IG1602 and RNAi feeding on this strain. ........................ 53 Figure 4.1 Immunoprecipitation of SapA-SapB complex............................................... 108 Figure 4.2 Spore morphology difference between ATCC 96282 and ARSEF 6962. ..... 110 Figure 4.3 PCR verification for the extra 10 kb sequence in ARSEF 6962 genome. .... 112 Figure 4.4 Feeding and washing method for sample preparation.. ................................. 114 Figure 4.5 Protein localization from the 2nd round of mass spectrometry analysis ........ 116 IV Figure 4.6 Peptide counts from a published APEX study. .............................................. 121 TABLE OF TABLES Table 1.1 NLP and CNC genes and their induction by different stimuli ............................ 3 Table 1.2 Typical infection structures of some nematophagous fungi .............................. 5 Table 1.3 Example of selected functionally verified fungal effectors in plant pathogenic fungi .................................................................................................................................. 14 Table 2.1 Plasmids constructed based on Gibson Assembly and ccdB selection ............. 36 Table 2.2 Transgenic strains obtained with the protocol of Article 1 ............................... 46 Table 3.1 Key RNAi proteins in D. coniospora ............................................................... 50 Table 4.1 Worm proteins found in JEM907-infected worms ......................................... 110 Table 4.2 Three rounds of Mass spectrometry analysis .................................................. 116 Table 4.3 Proteins from Mass spectrometry analysis ...................................................... 118 V CHAPTER 1. INTRODUCTION 1.1 Host-pathogen interactions 1.1.1 C. elegans and its innate immunity C. elegans is a small (around 1 mm in length), free-living worm, generally found in rotting fruit and plant matter. It feeds on bacteria and fungi. In the laboratory, it can be maintained on agar plates with a spread layer of Escherichia coli as its food. Starting in the mid-1960s, Sydney Brenner developed C. elegans as a model organism, for studying developmental biology and neurobiology (BRENNER 1974). It takes 3 days for the worm to develop from embryo to adult at 20 °C. During its adulthood, a single hermaphrodite can produce around 300 genetically-identical progeny. The hermaphrodite can also be fertilized by males. Males arise at a very low frequency, 0.1% under standard laboratory conditions. The life span of C. elegans is about 2-3 weeks on average at 20 °C. C. elegans was the first multicellular organism to be sequenced (THE C. ELEGANS SEQUENCING CONSORTIUM 1998). Strains are generally of the same, defined, genetic background. Loss- of function mutants for most genes are available. Additionally, one can knock-down most of its genes with RNAi by feeding (further introduced in 1.2.1.1). All these factors have made C. elegans a good model for various biological studies. Pioneered by Ausubel’s lab, C. elegans has also emerged as a model organism for the study of host-pathogen interactions in the last 15 years (MAHAJAN-MIKLOS et al. 1999; TAN et al. 1999). There are 3 layers for C. elegans defence against pathogens. Firstly, worms are capable of distinguishing some pathogens from non-pathogenic microbes and move away 1 from them, thus potentially avoiding infection (SCHULENBURG AND EWBANK 2007; MCMULLAN et al. 2012; NAKAD et al. 2016). In addition, there are two physical barriers