Comparative Genomic and Transcriptional Analyses of Magnaporthe oryzae and other Eukaryotes

Magnaporthe oryzae causes devastation of rice crops around the world; destroying enough to feed at least 60 million people annually. Here-in, I investigate this pathogenic fungus to elucidate the genomic components utilized during infection. Investigated components include regulatory elements, RNA sequences, and genes (especially transcription factors). Chapter 1 contains the background and introduction to my research. In Chapter 2, I characterize a common transcription factor component, the basic Helix-Loop-Helix (bHLH) domain, in M. oryzae and other fungi. Through phylogenetic analyses I identified 12 major groupings within Fungi; identifying conserved motifs and functions specific to each group. Several classification models were built to distinguish the 12 groups and elucidate the most discerning sites in the domain. These models were highly accurate and led to the identification of 12 highly discerning sites (1, 4, 6, 7, 8, 12, 15, 16, 19, 20, 50, and 53), which were incorporated into a set of rules to classify sequences into one of these 12 groups. Conservation of amino acid sites and phylogenetic analyses established that, like plant bHLH proteins, fungal bHLH containing proteins are most closely related to animal Group B. In Chapter 3, I assessed the bHLH domain across Plants, Animals, and Fungi to identify unique sequence characteristics pertaining to each Kingdom. Using classification models, I identified five essential amino acid sites that are highly characteristic of these Kingdoms. Hidden Markov Models, built on expertly aligned domains, were used with the classification models to identify and classify bHLH sequences from a marine environmental sample. Last, I created an online tool that can align, extract, and classify bHLH sequences. Next generation sequencing was used to perform a detailed examination and characterization of small RNA molecules from mycelia and appressoria in Chapter 4. In a collaborative project, my work showed that genomic features contributed differentially to the RNA sequence libraries. Mycelia RNAs were enriched for intergenic and repetitive elements while a higher proportion of appressoria RNAs were enriched for tRNA loci. Differential mapping of small RNAs to the 5’ and 3’ halves of mature tRNAs was also observed. This led to the identification of sites with post-transcriptional modification within tRNAs and showed a difference in that modification between the two tissues. In a second collaborative RNA study (Chapter 5), methylguanosine-capped and polyadenylated small RNAs (CPA-sRNAs) were sequenced with 454 technologies. My work showed that CPA-sRNAs mapped to rRNAs, tRNAs, snRNAs, transposable elements and intergenic regions. Where CPA-sRNAs were mapped to protein coding genes, they were predominately associated with transcriptional start and termination sites. Those proteins enriched for CPA-sRNAs, especially ribosomal encoding proteins, were positively correlated with gene expression. Finally, in Chapter 6, I designed a new comparative genomics software package (D-SynD) that can detect regions of syntenic DNA between multiple large genomes simultaneously. D-SynD requires no gene models and makes no assumptions with regards to gene order or orientation. Additionally, detected syntenic regions are statistically evaluated for significance. The software allows many user options, such as defining the preferred syntenic region size and complexity. D-SynD is released as an open-source software package for use in comparative genomic studies