| Sclerotinia sclerotiorum (Lib.) de Bary is a devastating fungal phytopathogen with a broad host range and global distribution. The resting structures produced by this fungus, sclerotia, are crucial for survival in harsh environments and further dissemination of the fungus when environmental conditions become conducive. Sclerotia can germinate as hyphae to initiate disease directly or germinate as fruiting bodies, apothecia, to produce forcibly-discharged ascospores that act as a dispersible inoculum source. To begin a molecular genetic dissection of sclerotial developmental regulation, the first gene I chose for investigation was Ss_ssp1. The protein encoded by this gene was previously described as the major storage protein present in mature sclerotia of S. sclerotiorum. I found that ssp1 transcripts specifically accumulated in all stages of sclerotial development with peak levels in stage IV sclerotia. In contrast with the sclerotia-restricted spatial accumulation of ssp1 transcripts, Ssp1 protein accumulation was detected in all sclerotial and apothecial stages. Immunolocalization suggests the release of Ssp1 from sclerotial protein bodies and the relocation to apothecia during carpogenic germination. Contrary to our original hypothesis, Ss_ssp1 deletion does not distinctively affect sclerotial development or carpogenic germination. However, the upregulation of the Ss_ssp1 paralog, Ss_ssp2 and another 16kDa major protein in deletion mutants indicates a possible functional redundancy and compensatory role for the Ss_Ssp1 homolog and other sclerotia-accumulating proteins. To comprehensively investigate genes involved in sclerotial development, transcriptome profiling during sclerotial initiation was conducted using a genomic, long oligomer microarray. When compared to gene expression during hyphal growth, 15% of the genes from the S. sclerotiorum genome were differentially expressed (up- or down-regulated) during sclerotial initiation. Additionally, 14% or the orphan ESTs examined are predicted to be newly discovered genes on the basis of my microarray analysis and annotation. The gene encoding a gamma-glutamyl transpeptidas (Ss_ggt) was one of the genes whose expression was markedly upregulated during sclerotial initiation by microarray analysis. Gene deletion mutant of Ss_ggt resulted in distinct morphological aberrations in sclerotial morphology. In mature dry sclerotia, the cortex layer was thickened and easily peeled away with the rind from the medulla. Sclerotia of the Ss_ggt deletion mutant failed to carpogenically germinate into apothecia due to an internal breakdown of the interior sclerotial tissue during the carpogenic germination incubation period. This phenotype is attributed to poor environmental protection of the medulla, allowing the cortex to easily be separated from the rind outerlayer. |
