Molecular characterization of a novel segmented dsRNA mycovirus and its association with hypovirulence of Fusarium graminearum. Dissertation A thesis submitted to the Fachbereich Biologie, Universität Hamburg for the degree of doctor rerum naturalium By Darissa Omar Bethlehem, Palestine Hamburg, 2011 17 December 2010 Mr. Omar Darissa Bramfelder Chaussee 9 22177 Hamburg, Germany RE: Review of thesis entitled “Molecular characterization of a novel segmented dsRNA mycovirus and its association with hypovirulence of Fusarium graminearum” by Mr. Omar Darissa I confirm that the thesis of Mr. Omar Darissa was reviewed by me, a native English speaker, for English language accuracy. The thesis is well written, and therefore I would recommend that the dissertation be accepted in its current form. During my 42 years as a professor, I have read many theses and Mr. Darissa’s thesis meets the standards for the University of Wisconsin-Madison. Phone numbers: University of Wisconsin: 608-262-1410 Home: 608-845-7717 ([email protected] or [email protected]) To my parents Mousa and Meriam Darissa To my wife Laila Darissa To my children Mousa, Mahmoud, and Mamoun Contents Contents Contents..................................................................................................................... i List of Figures ......................................................................................................... v List of Tables ......................................................................................................... vii ABBREVIATIONS .................................................................................................. viii 1. Introduction ..................................................................................................... 1 1.1. Mycoviruses .......................................................................................................... 1 1.1.1. dsRNA mycoviruses ........................................................................................... 2 1.1.1.1. Family Totiviridae ............................................................................................ 3 1.1.1.2. Family Partitiviridae ........................................................................................ 3 1.1.1.3. Family Chrysoviridae ...................................................................................... 4 1.1.1.4. Family Reoviridae ............................................................................................ 5 1.1.2. Positive-strand RNA mycoviruses ...................................................................... 6 1.1.3. DNA mycoviruses ............................................................................................... 6 1.2. Mycovirus associated hypovirulence. ................................................................... 6 1.3. Mycoviruses of F. graminearum ............................................................................ 8 1.4. Fusarium head blight ............................................................................................ 8 1.4.1. The fungus Fusarium graminearum .................................................................... 8 1.4.2. The disease cycle of F. graminearum in wheat. .................................................. 9 1.5. Methods for the sequence determination of dsRNA templates. ........................... 10 1.5.1. Random PCR (rPCR). ........................................................................................ 10 1.5.2. SPAT and FLAC methods. ................................................................................ 12 1.5.3. Direct cloning of dsRNA into dsDNA vectors. .................................................. 14 1.6. Aims of this study ............................................................................................... 15 2. Material and Methods ................................................................................ 16 2.1 Material ............................................................................................................. 16 2.1.1 Enzymes and chemicals .................................................................................... 16 2.1.2. Microbial strains and culture conditions. ......................................................... 16 2.1.3. Media and buffers ............................................................................................ 17 2.1.4. Oligonucleotides (primers)............................................................................... 18 2.2 Methods ............................................................................................................ 21 2.2.1 Isolation and purification of dsRNA .................................................................. 21 2.2.2 DNA extraction using the CTAB method ............................................................ 22 i Contents 2.2.3 Phenol extraction method of total nucleic acids ............................................... 22 2.2.3 Random PCR (rPCR) .......................................................................................... 22 2.2.4 Single Primer Amplification Technique (SPAT) ................................................. 23 2.2.5 Full length Amplification of cDNA (FLAC) ......................................................... 24 2.2.6 Direct ligation of dsRNA into pJET1.2 and pGEM ®-T vectors: ........................... 24 2.2.7 Cloning and sequencing: .................................................................................. 25 2.2.7.1 Preparation of electrocompetent cells. .......................................................... 25 2.2.7.2 Preparation of chemical competent cells. ...................................................... 25 2.2.7.3 Transformation of competent cells. ................................................................ 26 2.2.7.4 MiniPreps and restriction digestion. .............................................................. 26 2.2.8 Molecular identification of China 9 isolate. ....................................................... 26 2.2.9 Purification of Virus-Like Particles ................................................................... 26 2.2.9.1 Transmission Electron Microscope (TEM). ..................................................... 27 2.2.10. Hyper immunization of rabbits. ...................................................................... 27 2.2.11. Purification of the antibodies. ........................................................................ 28 2.2.12. Ultrastructural studies. ................................................................................. 28 2.2.12.1 Primary and secondary Fixations ................................................................. 28 2.2.12.2 Dehydration, infiltration, and embedding ..................................................... 28 2.2.12.3 Sectioning and TEM. ..................................................................................... 29 2.2.12.4 Immunohistology. ......................................................................................... 29 2.2.13 Northern Blot analysis. ................................................................................... 30 2.2.14 Southern Blot analysis. ................................................................................... 30 2.2.15 Protein sequence analysis. ............................................................................. 30 2.2.16 Labeling of virus surface proteins. ................................................................. 31 2.2.17 Western blot .................................................................................................... 31 2.2.18 Relative quantification PCR............................................................................. 31 2.2.19 Virulence assay on wheat heads. .................................................................... 32 2.2.20 Virulence assay on maize cobs. ...................................................................... 32 2.2.21 Growth assays ................................................................................................. 33 2.2.21.1 Production of Perithecia. .............................................................................. 33 2.2.21.2 Transmission of FgV-ch 9 through conidia ................................................... 33 2.2.21.2.1 Reverse transcription: ............................................................................... 33 2.2.21.2.2 PCR: .......................................................................................................... 34 2.2.22 Expression of FgV-ch9 in F. graminearum PH-1 .............................................. 34 2.2.22.1 Semi-quantitative PCR. ................................................................................. 35 2.2.23 Dicer 2 gene disruption by double homologous recombination ...................... 35 2.2.24. Preparation of F. graminearum protoplasts. ................................................... 35 2.2.24.1. Transformation of F. graminearum protoplasts with plasmid constructs. ... 36 ii Contents 2.2.24.2 Protoplast transfection with purified VLPs. .................................................. 36 2.2.25 Data analysis and accession numbers. ........................................................... 37 3. Results ............................................................................................................. 38 3.1. Optimization of the methods for the sequence-determination of dsRNA templates.