University of Dundee DOCTOR OF PHILOSOPHY Molecular characterisation of the Rhynchosporium commune interaction with barley Gamble, Mary Award date: 2016 Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 05. Oct. 2021 Molecular characterisation of the Rhynchosporium commune interaction with barley Louise Gamble Thesis submitted for the degree of Doctor of Philosophy University of Dundee September 2016 I Contents CONTENTS II LIST OF FIGURES VII LIST OF TABLES X LIST OF ABBREVIATIONS__________________________________________ XI ACKNOWLEDGEMENTS XIV DECLARATION XV STATEMENT XV ABSTRACT XVI 1. GENERAL INTRODUCTION _______ 1 1.1 Economic importance of R. commune infection on barley 1 1.2 Introduction to R. commune 2 1.2.1 Origin and host specialisation of R. commune 2 1.2.2 Classification 4 1.2.3 Epidemiology 7 1.2.4 Infection process and disease symptoms 9 1.2.5 R. commune in the plant apoplast 12 1.3 Pathogen - Host Interactions 12 1.3.1Evolutionary basis of host-pathogen interactions 12 1.3.2 Effectors and their role in disease 17 1.3.3 R. commune effectors – Necrosis Inducing Peptides 19 1.3.4 AvrRrs1 – Rrs1 interaction 22 1.4 Barley resistance to R commune 23 1.4.1Major R gene and partial resistance 23 1.4.2Molecules involved in a resistant response 24 1.5 Pathogen evasion of plant defence 25 1.6 Scope of this thesis 27 2. MATERIALS AND METHODS 29 2.1 Plant seedling growth 29 2.2 Culturing and storage of micro-organisms 2.2.1 Fungi 29 2.2.2 Bacteria 30 II 2.2.3 Harvesting of fungal spores 30 2.3. R. commune infection of barley 30 2.3.1. Detached leaf assay 30 2.3.2 Infection time course 31 2.4 Microscopy 32 2.4.1 Lacto phenol trypan blue staining 32 2.4.2 Sample preparation for confocal microscopy 32 2.4.3 Confocal laser scanning microscopy (CLSM) 32 2.5 Gene expression 33 2.5.1 Total RNA, mRNA extraction and cDNA synthesis 33 2.5.2 Real-time RT-PCR (qRT-PCR) 33 2.6 Cloning strategy and expression plasmids construction 34 2.6.1 Preparation of competent yeast cells 34 2.6.1 Preparation of S. cerevisiae competent cells 35 2.6.2 P. pastoris electro competent cells 35 2.6.3 Yeast re-combinational cloning (YRC) 35 2.6.4 Plasmids used for YRC 36 2.6.4.1 Cloning plasmid 36 2.6.4.2 Expression plasmids 36 2.7 Transformation protocols 36 2.7.1 E. coli transformation 36 2.7.2 S. cerevisiae transformation 37 2.7.3 P. pastoris transformation 38 2.7.4 Electroporation transformation of R. commune 38 2.7.5 Transformation of fungal protoplasts 39 2.7.5.1 Protoplast preparation 39 2.7.5.2 Protoplast Transformation 39 2.7.6 Screening of yeast transformants by colony PCR 41 2.7.7 Protein expression analysis in P. pastoris cultures 41 2.7.1 Protein concentration 41 2.7.2 Protein purification 41 2.8 Molecular biology protocols 42 2.8.1 Fungal & plant DNA extractions 42 2.8.2 Plasmid extraction 42 2.8.2.1 Plasmid extraction from E. coli 42 2.8.2.2 Plasmid extraction from yeast cells 42 2.8.2 Polymerase chain reaction 43 2.9 Proteomics protocols 44 2.9.1 Protein extraction 44 2.9.2 Protein visualisation 45 2.9.2.1 SDS-polyacrylamide-gel electrophoresis 45 2.9.1.2 Western blotting 45 2.9.3 Proteome analysis of barley apoplast 46 2.9.3.1 Apoplastic fluid extraction of barley leaves 46 2.9.3.2 Enzyme contamination assay 46 2.9.3.3 Sample preparation for MS analysis: In gel digestion 47 2.9.3.4 Sample preparation for MS analysis: In Solution Digestion 48 2.9.3.5 Sample cleaning using C18 Ziptip columns 48 2.9.3.7 Dimethyl labelling procedure 49 III 2.10 Polysaccharide binding assay 50 2.11 In Vitro Fungal Growth Assays 50 2.12 Bioinformatic analysis 51 2.12.1 Sequence analysis tools 51 2.12.2 Proteomic analysis 52 2.12.3 Genome databses 52 3 IDENTIFICATION AND CHARACTERISATION OF R. COMMUNE CANDIDATE EFFECTOR GENES 53 3.1 Introduction 53 3.2 Results 55 3.2.1 Background – Bioinformatics pipeline reveals a plethora of candidate R. commune effectors 55 3.2.2 Candidate effector sequence analysis 57 3.2.3 Homology of R. commune candidate effectors to other fungal proteins 57 3.2.4 R. commune candidate effector gene expression in planta and in vitro 60 3.2.4.1 Analysis of candidate effector expression in R. commune strain L2A during infection in planta 60 3.2.4.2 Analysis of expression of selected candidate effectors during infection in planta and culture media using two different R. commune strains 64 3.2.5 R. commune transformation to elucidate gene function 70 3.2.6 Analysis of the apoplastic proteome during infection reveals the presence of potential effectors 73 3.2.6.1 Enzyme contamination assay 73 3.2.6.2 R. commune proteins identified in the apoplast 74 3.3 Discussion 80 4 IDENTIFICATION AND CHARACTERISATION OF A FAMILY OF LYSM- DOMAIN PROTEINS 84 4.1 Introduction 84 4.2 Results 87 4.2.1 A large family of LysM domain genes identified in the R. commune genome 87 4.2.2 Phylogenetic analysis of LysM domains from members of R. commune 92 LysM domain protein family 4.2.3 Expression analysis of genes coding for LysM domain proteins reveals upregulation during infection of barley 95 4.2.4 RcLysM3 protein shares sequence homology with Z. tritici Mg3LysM and C. fulvum Ecp6 102 4.2.5 In vitro analysis confirms the ability of RcLysM3 to bind chitin 103 4.2.6 RcLysM3 does not provide protection against plant hydrolytic enzymes 106 4.2.7 Correlation between virulence/avirulence of R. commune strains on barley cultivar La Mesita and a SNP change in the RcLysM3 allele 107 4.3 Discussion 109 IV 5 ANALYSIS OF BARLEY RESISTANCE TO R. COMMUNE_____________114 5.1 Introduction 114 5.2 Results 117 5.2.1 Rrs3, Rrs4 and Rrs13 show a high level of resistance to R. commune isolates 117 5.2.2 Analysis of asymptomatic infection on barley lines using R. commune strain 119 214-GFP 5.2.3 Analysis of asymptomatic infection of barley landraces using R. commune 122 strain 214-GFP 5.2.4 Quantitative proteomics 124 5.2.4.1 Confirmation of barley response to R. commune during infection 124 5.2.4.2 Sequence analysis of defence related proteins identified in barley 126 apoplastic proteome 5.4.5.3 Analysis of protein expression reveals upregulation of (1-3) 129 β-glucanase and α-L arabinofuranosidase in Atlas 46 at 4 dpi 5.3 Discussion 133 6 CHARACTERISATION OF R. COMMUNE INTERACTION WITH N. BENTHAMIANA 136 6.1 Introduction 136 6.2 Results 139 6.2.1 Asymptomatic growth of R. commune does occur on the model plant species N. benthamiana 139 6.2.3 Growth of R. commune strain 214-GFP on non-plant surfaces 143 6.2.4 R. commune gene expression during growth on non-host N benthamiana 144 6.2.4.1 Analysis of NIP expression on non-host N benthamiana 144 6.2.4.2 Analysis of expression during growth on N. benthamiana leaves of the R. commune effector RcINS1 inducing cell death in N. benthamiana. 147 6.3 Discussion 148 7 GENERAL DISCUSSION 151 8 REFERENCES 158 9 APPENDIX 181 9.1 Liquid and agar media 181 9.1.2 CZV8CM agar 181 9.1.2 Potato Dextrose Broth / Agar (PDB /PDA) 181 9.1.3 Yeast Peptone Dextrose (YPD) 181 9.1.4 YNB + glucose 182 9.1.5 Luria Broth and Agar plates (LB) 182 9.1.6. Synthetic complete minus uracil (SC-Ura) 182 9.1.7 Low salt Luria broth (LB) 182 9.1.8 SOC media 183 9.2 Lacto phenol trypan blue solution 183 9.2.1 Staining solution 183 V 9.2.2. De-staining solution 184 9.3 Solutions and reagents for transformation 184 9.3.1 Protoplast preparation 184 9.3.1.1. KC solution 184 9.3.1.2. MT solution 184 9.3.1.3. KC-MT solution 184 9.3.1.4 Protoplasting enzymes 185 9.3.1.5 PEG solution 185 9.3.2 Yeast transformations 185 9.3.2.1 SORB solution 185 9.3.2.2 PEG/LiAc 185 9.3.2.3 TE buffer 186 9.3.2.4 STET buffer 186 9.4 Solutions and reagents for proteomic experiments 186 9.4.1 Protein extraction buffer 186 9.4.2 PBS buffer 187 9.4.3 Blocking solution 187 9.4.4 Bead elution buffer 187 9.4.5 HNT buffer 187 9.4.6 HN buffer 188 9.4.7 Trypsin solution 188 9.5 List of intensity values for plant proteins identified in the apoplastic fluid of 189 infected and non-infected barley leaves at 4 and 7 dpi 9.6 List of proteins identified in the apoplastic fluid from cultivars Optic, Atlas 195 and Atlas46, infected and non-infected leaf material VI List of figures Figure Title Page 1.1 Spores of Rhynchosporium commune and barley cultivar Optic infected with R.