Development of silencing vectors for Aspergillus fumigatus based on mycoviruses and short interspersed nuclear elements LAKKHANA KANHAYUWA A thesis submitted for the degree of Doctor of Philosophy and the Diploma of Membership of Imperial College Imperial College London Department of Life Sciences Division of Cell and Molecular Biology 1 Declaration of Originality I hereby declare that this thesis is the product of my own work unless otherwise acknowledged in the text or by references and has not been previously submitted for a degree in any university or other purposes. Signed: Lakkhana Kanhayuwa Date: September, 2015 2 Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. Abstract 3 Abstract Title of Thesis: Development of silencing vectors for Aspergillus fumigatus based on mycoviruses and short interspersed nuclear elements. A novel mycovirus named Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) was discovered and characterized in the human pathogenic fungus, A. fumigatus clinical isolate Af293. The virus reveals several unique features not previously found in double-stranded RNA (dsRNA) viruses and represents the first dsRNA that is infectious both as a purified entity and a naked dsRNA. The AfuTmV-1 is an unencapsidated dsRNA mycovirus comprised of four genomic segments, ranging in size from 1.1 to 2.4 kbp. The largest component encodes a putative viral RNA-dependent RNA polymerase (RdRP) where the sequence of the most highly conserved motif changes from GDDX to GDNQ. The third largest dsRNA encodes an S-adenosyl methionine-dependent methyltransferase (SAM) capping enzyme and the smallest dsRNA encodes a proline-alanine rich protein. Short interspersed nuclear elements (SINEs) were also identified in the fungal genome. Identification of the elements revealed tRNA-related and 5S rRNA-related SINE families which showed variation in transcription activity and copy number. AfuTmV-1 sequences together with SINEs were subsequently exploited to develop alternative tools for silencing genes in A. fumigatus. A truncated AfuTmV-1 based vector was successfully constructed and used as a prototype vector for generating a recombinant virus-induced gene silencing (VIGS) vector. Transcriptional fusion SINE-derived vectors were also developed to silence an ALB1/PKSP gene responsible for conidial pigmentation. With anticipation that the development will provide a powerful reverse genetic tool for functional genomics studies to identify key elements involved in fungal pathogenicity and also provide a medical benefit in exploiting mycoviruses as a future therapeutic agent against fungal infections. RNA silencing | mycovirus | SINEs | Aspergillus fumigatus Table of Contents 4 Table of Contents Abstract I 3 Table of contents 4 List of Figures 10 List of Tables 15 Abbreviations 17 Introduction and project objectives 23 Chapter 1: Literature review 27 1. Mycoviruses 28 1.1 General characteristics of mycoviruses 28 1.1.1 Double-stranded RNA (dsRNA) mycoviruses 29 1.1.2 Replication of dsRNA mycoviruses 29 …………..1.1.3 Mode of transmission 31 1.1.4 Effect of mycovirus on host phenotype 32 1.2 Mycoviruses in Aspergillus spp. and other human pathogenic fungi 34 1.3 Self-defense mechanisms in fungi and mycoviruses 36 1.3.1 Antiviral defence mechanisms in fungi 36 1.3.2 Antifungal defence mechanisms in mycoviruses 37 1.4 Application of mycoviruses 38 …………..1.4.1 Application of mycoviruses in agriculture 38 …………..1.4.2 Application of mycoviruses in industry 39 …………..1.4.3 Application of mycoviruses in medicine 39 2. Aspergillus fumigatus 40 2.1 General characteristics and genomic information 40 2.2 Pathogenicity and genes involving fungal virulence 41 2.3 Treatment of invasive aspergillosis 43 3. Transposable elements (TEs) in fungi 44 3.1 Retrotransposable elements (Retrotransposons) 45 Table of Contents 5 3.1.1 LTR retrotransposons 45 3.1.2 Non-LTR retrotransposons 45 3.1.2.1 Long interspersed nuclear elements (LINEs) 46 3.1.2.2 Short interspersed nuclear elements (SINEs) 47 3.2 Retrotransposons in A. fumigatus 53 3.3 Application of SINEs 54 3.3.1 Phylogenetic marker and genomic tag 54 3.3.2 Regulation of gene expression 55 4. RNA silencing in fungi 55 4.1 Cellular mechanism of RNA silencing 55 4.2 RNA silencing machinery in fungi 57 4.3 RNA silencing in fungi in response to virus infection 58 4.4 Application of RNA silencing 59 5. Development of gene silencing approaches in fungi 61 5.1 Direct delivery of siRNA/dsRNA 61 5.2 Host-induced gene silencing vectors (HIGS) 62 5.2.1 Short hairpin RNA (shRNA) expressing/silencing plasmid vectors 62 5.2.2 Bidirectional-dual promoter system 63 5.3 Virus-induced gene silencing vectors (VIGS) 63 Chapter 2: Materials and Methods 66 1. Fungal maintenance and harvesting 67 2. Nucleic acid manipulation 67 2.1 Extraction of A. fumigatus genomic materials for small scale preparation 67 2.2 Extraction of A. fumigatus total RNA for large scale preparation 68 2.3 Extraction of viral dsRNA (LiCl fractionation) 69 2.4 Isolation of mycovirus particles 70 2.5 Phenol: Chloroform extraction 71 2.6 Purification of viral dsRNAs 71 2.7 Purification of A. fumigatus total RNA 72 2.8 Nucleic acid precipitation 72 2.9 Agarose gel electrophoresis 73 2.10 Recovery of the nucleic acid samples from agarose gels 73 Table of Contents 6 2.11 Protein analysis by SDS-PAGE 73 2.12 In-gel protein digestion 74 3. Construction of cDNA library from dsRNA by random-PCR (rPCR) 75 4. Genome walking strategy 77 4.1 Primer design 77 4.2 Synthesis of cDNA from dsRNA by genome walking 77 4.3 RNA linker-mediated rapid amplification of cDNA ends (RLM-RACE) 78 5. Cloning techniques 80 5.1 Ligation 80 5.2 Preparation of Escherichia coli JM 109 competent cells 81 5.3 Transformation of plasmids into E. coli 81 5.4 Screening of the transformants 82 5.5 Restriction enzyme digestion 82 5.6 Sequencing and phylogenetic analyses 83 6. Northern blot hybridisation of the mycovirus, AfuTmV-1 dsRNAs 84 6.1 In vitro transcription-labeling of RNA with digoxigenin 84 6.2 DIG northern blot hybridisation 86 7. Curing of A. fumigatus Af293 from AfuTmV-1 infection and effects of virus on fungal growth and morphology 88 7.1 Curing of A. fumigatus Af293 NK isolate with cycloheximide 88 7.2 Effects of dsRNA on growth of A. fumigatus Af293 NK isolate 88 8. In vitro replication of AfuTmV-1 89 9. In vitro translation of AfuTmV-1 dsRNAs 90 10. Transfection assay 91 10.1 Protoplast preparation 91 10.2 Transfection assay 92 11. Identification of SINEs in A. fumigatus Af293 genome 93 11.1 Sequence dataset 93 11.2 Search strategy and computational analyses 93 11.3 PCR amplification of A. fumigatus SINEs 95 Table of Contents 7 11.4 RT-PCR amplification of A. fumigatus SINEs 96 11.5 Southern blot hybridization of A. fumigatus SINEs 97 11.6 Detection of small RNA homologous to SINEs 99 11.7 Secondary structure prediction of A. fumigatus SINE transcripts 101 12. Silencing vector construction 102 12.1 Construction of silencing vectors based on AfuTmV-1 dsRNA mycovirus 102 12.2 Construction of silencing vectors based on a SINE element using fusion PCR 104 12.3 Transfection and transformation assays for gene silencing study 107 Chapter 3: Results and Discussion (Part I) 110 1. Characterisation of Aspergillus foetidus virus-fast dsRNA4 (AfV-F dsRNA4) 111 1.1 Construction of a cDNA library of AfV-F dsRNA4 by rPCR method 112 1.2 Determination 5’-and 3’- termini of AfV-F dsRNA4 by RLM RACE 112 1.3 The genomic sequence of AfV-F dsRNA4 113 1.4 Characterisation of the defective RNA of A. foetidus virus-slow (AfV-S) 116 Chapter 3: Results and Discussion (Part II) 117 Abstract II 118 2. Characterization of Aspergillus fumigatus tatramycovirus (AfuTmV-1) 119 2.1 Sensitivity of AfuTmV-1 and dsRNA to DNase, S1 and RNaseIII 120 2.2 Construction of cDNA clone library of AfuTmV-1 dsRNAs by rPCR 121 2.3 Determination of the 5’- and 3’-termini of dsRNA2 using RLM-RACE 124 2.4 The genomic sequence of AfuTmV-1 dsRNA2 125 2.5 Construction of a cDNA clone library of dsRNA1 by rPCR and RLM-RACE 129 2.6 The genomic sequence of AfuTmV-1 dsRNA1 130 2.7 Construction of a cDNA clone library of dsRNA3 by rPCR and RLM-RACE 132 2.8 The genomic sequence of AfuTmV-1 dsRNA3 133 2.9 Determination of the 5’- and 3’-termini of dsRNA4 using RLM-RACE 137 2.10 The genomic sequence of AfuTmV-1 dsRNA4 138 2.11 Multiple nucleotide sequences alignment of AfuTmV-1 dsRNAs 143 2.12 A DNA copy of AfuTmV-1 does not reside in the Af293 genome 145 2.13 Phylogenetic analysis of the putative RdRP of AfuTmV-1 146 2.14 Northern blot analysis of AfuTmV-1 dsRNAs from Af293 isolate 158 2.15 Purification of the AfuTmV-1 VLP and SDS-PAGE analysis 158 Table of Contents 8 2.16 Quantitation of AfuTmV-1 dsRNAs and ssRNAs in infected mycelia 161 2.17 In vitro replication of the AfuTmV-1 dsRNAs 164 2.18 In vitro translation of the AfuTmV-1 dsRNAs 164 3. Biological comparison of virus-cured and virus-infected fungal strains 166 3.1 Curing A.