Structural, Functional and Evolutionary Analyses of the Rice Blast fungal Genome

Abstract

Disease caused by rice blast results in a dramatic loss of rice production worldwide every year. The rice blast fungus, Magnaporthe oryzae, has long been the subject of intense investigation and has been adopted as a model system to elucidate molecular host-pathogen interactions. In recent years, a major initiative to decode the genome of this fungus was launched, which has greatly shaped current biological studies. This dissertation contains three projects developed to help with genome sequencing as well as analyses to explore genome function and evolution. The first project involved functional characterization of ACI1. This gene was identified in a yeast two-hybrid experiment to interact with MAC1, a key regulator of the infection process. However, deletion of ACI1 had no obvious effect on pathogenesis. The second project validated and refined the previously constructed physical map of chromosome 7 in preparation for a map based sequencing project. One hundred and forty out of the 155 non-repetitive DNA-containing ESTs were confirmed to be chromosome 7 anchored. Furthermore, 5 previously unanchored FPC contigs were anchored to chromosome 7. Following the release of the whole genome sequence and other related fungi, an evolutionary analysis of the cytochrome P450 superfamily was undertaken as a third project. Cytochrome P450s form a large protein superfamily, representing ˜ 1% of predicted ORFs and are present in many organisms from bacteria to human. The functions of P450 enzymes are very varied. In fungi, they are involved in detoxifying plant defense compounds as well as being required for biosynthesis of pathogenesis related toxins. Two plant pathogenic fungi Fusarium graminearum and M. oryzea with two saprophytic fungi Aspergillus nidulans and Neurospora crassa were chosen to compare their P450omes. Due to considerable sequence divergence that hampered application of standard phylogenetic approaches, a novel approach was developed to reconstruct their evolution. The results suggested that the ancestral genome of the four fungi contained similar number of P450s as found in current filamentous ascomycetes. As a general trend, during evolution, new lineage specific P450 have appeared with a corresponding loss of genes from ancestral clans. M. oryzae exhibited a larger expansion with less contraction compared to the other three fungi. In particular genes were expanded or maintained in two P450 clans that may be involved in the alkane and fatty acid assimilation.