UBC Theses and Dissertations
Genetic dissection of Sclerotinia sclerotiorum biology using forward genetics Xu, Yan
Diverse plant fungal pathogens that cause worldwide crop losses are understudied due to various technical challenges. With the increasing availability of sequenced whole genomes of these non-model fungi, effective genetic analysis methods are highly desirable. My PhD projects focus on the establishment of a new gene discovery pipeline, which combines a fast forward genetic screening in large scale with high-throughput next-generation sequencing. I applied this pipeline in the notorious soilborne phytopathogen, Sclerotinia sclerotiorum, and identified 32 mutants with various developmental and growth deficiencies. Detailed molecular studies of three melanisation-deficient mutants provide a proof of concept for the effectiveness of our method. Furthermore, using this forward genetic approach, a RAS-GTPase activating protein, SsGAP1, was identified, which plays essential roles in sclerotia formation, compound appressoria production and virulence. To find the responsible RAS-GTPase being activated by SsGAP1 through mutant phenotypes comparison, I knocked down two RAS-GTPases, SsRAS1 and SsRAS2, respectively. My results revealed that both SsRAS1 and SsRAS2 are required for vegetative growth, sclerotia formation, compound appressoria production and virulence. As RNA can move from plant hosts to associating eukaryotic pathogens, trans-species RNAi pathway has been employed for pathogen control through host-induced gene silencing (HIGS). Thereby, I tested whether these RAS signaling genes can be used as HIGS targets to control stem rot caused by S. scleortiorum. Here, when I infiltrated tobacco leaves with Agrobacterium with RNAi constructs targeting SsGAP1 and SsRAS1, I observed reduced virulence. Taken together, my forward genetics gene discovery pipeline in S. sclerotiorum is highly effective in identifying novel HIGS targets to control fungal diseases caused by S. sclerotiorum. As RAS signaling is conserved in eukaryotes, these RAS signaling genes may also have the potential to be used for controlling other fungal diseases in higher plants. Altogether, with the successful identification of these causal genes responsible for the phenotypes of their respective mutants, it is foreseeable that this pipeline can be applied to facilitate efficient in-depth studies of other non-model fungal species and identify more novel HIGS targets against fungal pathogens in the future.
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