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Gong, Yihan

University of British Columbia

Sclerotinia sclerotiorum is a devastating fungal disease that contributes to significant crop yield losses worldwide. Strategies have been developed to combat this pathogen from propagation, however, most of them remain rather ineffective largely due to the formation of sclerotia, which are highly compacted mycelial masses covered by melanized protective outer layer. Prior studies have elucidated multiple signaling pathways involved in sclerotia development, and oxalic acid was identified as its major pathogenicity factor. In this thesis, using a previously established pipeline for rapid gene discovery in S. sclerotiorum, a forward genetic screen was carried out in an oxalic acid-deficient background, or 4.4 background, in search of additional regulators of sclerotia formation and oxalic acid-independent pathogenicity factors. In total, 7000 colonies were screened for non-sclerotia forming phenotypes, which yielded 7 putative mutants that exhibit different extents of sclerotia-forming deficiency. Next-generation sequencing of mutants’ genomic DNA and bioinformatic analyses were performed in an attempt to identify the causal mutation of each mutant. Three mutants have their candidate gene lists generated in the current library. In addition, two autophagy-related genes, SsAtg4 and SsAtg9, were identified to be the causal mutations of two mutants from the previous forward genetic screen in WT background. Knocking out these genes in S. sclerotiorum protoplasts through homologous recombination resulted in two deletion lines for each gene. Phenotypic analyses of these lines suggested that both SsAtg4 and SsAtg9 are required for sclerotia formation in S. sclerotiorum. However, other developmental processes, including compound appressoria formation and pathogenicity, appeared to be unaffected when these genes were disrupted. Taken together, two additional regulators of autophagy were identified to be required for sclerotia formation. Collectively, my M.Sc. thesis is an application of a gene discovery pipeline for identification of additional sclerotia-forming regulators and pathogenicity factors, which contributes to a better understanding of sclerotia formation in S. sclerotiorum.

Text

2025-05-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88058

Botany

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/