Elucidating the Repertoire and Biogenesis of Small RNAs in the Rice Blast Fungus, Magnaporthe oryzae

Remarkable progress has been made in the development of host-induced gene silencing (HIGS) for further application in plant resistance against parasites. Most of the available knowledge on HIGS is towards silencing of plant pathogenic viruses. Recently, new findings suggest RNA movement from plant to fungal cells results in specific silencing of fungal transcripts. This finding provides great potential for future development and application of HIGS to control plant pathogenic fungi including Magnaporthe oryza e, the blast disease causing agent.;In chapter 1, I summarize important aspects of M. oryzae as a model plant pathogen. Extensive genomics and transcriptome resources are available for this fungus as well as forward and reverse genetics, which greatly facilitates post-genomics studies. Although significant progress has been made for understanding of the molecular mechanisms of the rice--- M. oryzae interaction, this fungus remains one of the major concerns for rice production worldwide and wheat blast is emerging as a potential risk for wheat producing areas.;In chapters 2 and 3, I present overviews of the importance of recent discoveries regarding small RNA and gene silencing in the context of new opportunities to explore host-fungal pathogen interactions and discuss this topic as two review chapters. In chapter 2, I summarize the functional RNA silencing protein machinery in plant pathogenic fungi and discuss strategies to employ RNA silencing for investigating the basis of fungal pathogenesis and controlling fungal diseases through HIGS. In Chapter 3, I review different classes of fungal small RNAs and describe new methodologies for characterization of small RNA and RNA silencing in plant pathogenic fungi. The concept of HIGS to control fungal diseases holds great promise, but much remains to be learned. I conclude both chapters highlighting fundamental questions that remain to be addressed.;M. oryzae is known to have functional RNA silencing protein machinery, however, knowledge of small RNAs in this fungus is lacking. In chapter 4, I employed next generation sequencing to perform a detailed examination and characterization small RNA molecules (15--40 nucleotides long) from mycelia and appressoria tissues. In mycelia tissue, small RNAs of the size 18--23 nt were enriched from intergenic and repetitive elements while appressoria contained a greater proportion of small RNAs of 28--35 nt mapping to tRNA loci. I named LTR retrotransposon-siRNAs (LTR-siRNA) and tRNA-derived RNA fragments (tRFs) for small RNAs mapping to repeats in the mycelia and to tRNA in the appressoria libraries, respectively. Although their exact roles remain to be elucidated, the observed differential small RNA profiles suggest these molecules may play important roles in genome defense and regulation of growth and development in a tissue specialized manner.;In chapter 5, I investigate the possible roles of two T2 ribonucleases (MoRNS2-1 and MoRNS2-2) in the biogenesis of tRFs. MoRNS2-1 gene deletion mutants were obtained and were similar to wild type and ectopic strains in respect to conidiation, appressorium formation, vegetative growth and ability to cause disease in barley leaves. In addition, the tRFs profile was indistinguishable between deletion mutants and control strains. In contrast, I was unable to obtain gene deletion mutant of MoRNS2-2. Further characterization of MoRNS2-2 is required before I can conclude that MoRNS2-1 plays no role in biogenesis of tRFs or other small RNA classes.