Date of Graduation

12-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Plant Science (PhD)

Degree Level

Graduate

Department

Plant Pathology

Advisor

Burton Bluhm

Committee Member

Frederick Spiegel

Second Committee Member

Won-Bo Shim

Third Committee Member

Michael Evans

Fourth Committee Member

James Correll

Abstract

Gray leaf spot, caused by Cercospora zeae-maydis, is a devastating disease of maize that reduces yields and increases management costs. C. zeae-maydis penetrates maize leaves through stomata, but the biological and molecular bases of this process are poorly understood. The goal of this research was to elucidate the biological parameters of stomatal infection in C. zeae-maydis, and to identify and characterize novel genetic pathways involved in stomatal sensing and pathogenesis. Histopathological observations of a GFP-expressing strain of C. zeae-maydis during infection of maize indicated that the fungus responded to host-derived stomatal cues during the infection process. C. zeae-maydis was observed exhibiting tropism toward non-host stomata, which ultimately implicated molecular oxygen as a possible stomatal chemoattractant. To explore the role of circadian rhythmicity in gray leaf spot, the putative central circadian oscillator gene FRQ in C. zeae-maydis was functionally disrupted and characterized. Interestingly, FRQ deletion strains were non-pathogenic when inoculated on maize leaves. Histological observations suggested that FRQ deletion strains failed to form appressoria in association with maize stomata. In order to identify other novel genes involved in pathogenesis, a collection of 1228 insertional mutants was created and assayed for infectious development. Ten mutants were identified, including one that was disrupted in RJP1, a putative epigenetic regulator of gene expression. In a related study, thirty-one genes were selected for functional disruption based on sequence similarity to known fungal regulatory genes. Analysis of these mutants indicated that GPA2, which encodes a putative G protein alpha subunit, was required for pathogenesis. Lastly, gene expression analysis during pre-penetration infectious development revealed widespread transcriptional reprogramming. Key findings from this research include the discovery of novel pathogenesis-related genes and potential roles for oxygen sensing and the fungal circadian clock in foliar pathogenesis. Furthermore, this research illuminated a previously unrecognized level of complexity underlying the regulation of stomatal infection during gray leaf spot of maize and established a foundation for future molecular investigations. 

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