Testing the effect of in planta RNA silencing on Plasmodiophorabrassicae infection A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University S. R. Bulman 2006 ii Contents Abstract. ...................................................................................................., ................ v Acknowledgements .................................................................................................. vii List of Tables ........................................................................................................... viii List of Figures ........................................................................................................... ix CHAPTER 1. Literature Review ................................................................................ 1 Plasmodiophora brassicae ............................................................................................. 1 RNA silencing .................................................................................................................. 5 Plant defence by RNA silencing .................................................................................... 8 RNA silencing versus P. brassicae ............................................................................. 10 Molecular biology of P. brassicae ................................................................................ 10 Choice of cDNA cloning strategy in P. brassicae ....................................................... 12 Overall strategy ............................................................................................................. 14 CHAPTER 2. Identification of genes from the obligate intracellular plant pathogen, Plasmodiophora brassicae ....................................................................................... 15 Abstract .......................................................................................................................... 15 Introduction .................................................................................................................... 15 Materials and methods ................................................................................................. 17 Results ........................................................................................................................... 19 CHAPTER 3. Genomic DNA sequences from Plasmodiophora brassicae ............... 28 Abstract ..........................................................................................................................28 Introduction ....................................................................................................................28 Materials and methods .................................................................................................29 Results ...........................................................................................................................32 Discussion .....................................................................................................................37 CHAPTER 4. Testing plant-derived RNA silencing against the intracellular pathogen Plasmodiophora brassicae ....................................................................................... 42 Abstract ..........................................................................................................................42 Introduction ....................................................................................................................42 Material and methods ...................................................................................................44 Results .....................................................................................................; ..................... 47 Discussion ........................................................ ;............................................................. 51 CHAPTER 5. Discussion ......................................................................................... 54 Summary of research findings ..................................................................................... 54 SSH cDNA cloning techniques ....................................................................................55 Full-length cDNA cloning techniques .......................................................................... 56 iii The future of P. brassicae cDNA cloning .................................................................... 57 Genomic sequences from P. brassicae by PCR-walking .......................................... 57 Multiple P. brassicae actin genes ................................................................................58 New details of the P. brassicae genome .................................................................... 59 Evidence for RNA silencing machinery in plasmodiophorids? .................................. 59 Assessment of P. brassicae infection and choice of first hpRNA target .................. 60 Design of actin hpRNA construct.. ............................................................................... 61 Assessment of hpRNA expression .............................................................................. 62 In planta siRNAs showed no effect on P. brassicae infection ................................... 63 Why might RNA silencing have failed to be induced? ............................................... 63 Future research .............................................................................................................64 Literature ........- ..........................................................................................................66 Appendices ..............................................................................................................85 Appendix 1. Identification of genes from the obligate intracellular plant pathogen, Plasmodiophora brassicae ........................................................................................... 85 Quantitative RT-PCR analysis of P. brassicae gene expression .............................. 89 Appendix 2. Genomic DNA sequences from Plasmodiophora brassicae ............... 92 Degenerate PCR for Dicer proteins ............................................................................. 96 Appendix 3. Testing plant-derived RNA Silencing against the intracellular pathogen Plasmodiophora brassicae ... ........................................................................................ 99 Reporter-gene expression in P. brassicae galls ....................................................... 104 iv Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy Testing the effect of in planta RNA silencing on Plasmodiophora brassicae infection S. R. Bulman 2006 In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silenCing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting materia-I for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR­ walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain Signal peptides for ER translocation. v In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using peR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs. Keywords: Plasmodiophora