The development of RNA interference-based technologies for the control of Sclerotinia sclerotiorum in Brassica napus
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Date
2022-03-29
Authors
Wytinck, Nick
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Abstract
Necrotrophic fungal phytopathogens such as Sclerotinia sclerotiorum are a detriment to many economically significant crop species like canola, Brassica napus, and threaten the ability of the food supply chain to accommodate rising demand. Traditional approaches to control fungal pathogens predominantly involve chemical control through broad-spectrum agents that have been shown to negatively impact the agroecological environment. RNA interference (RNAi), an innate cellular pathway for the targeted silencing of mRNA molecules, may prove to be an effective alternative. I have investigated the utility of this technology both in the application of exogenous double stranded (ds)RNA (spray induced gene silencing – SIGS) to target specific mRNAs of S. sclerotiorum as well as through the creation of transgenic B. napus to express dsRNA molecules (host induced gene silencing – HIGS). I first investigated the mechanism in which S. sclerotiorum uptakes dsRNA. I performed microscopy using fluorescently labeled dsRNA and transgenic S. sclerotiorum expressing eGFP as well as molecular experiments using chemical inhibitors of cellular pathways and dsRNAs targeting the expression of specific genes within these pathways. Ultimately, I found clathrin mediated endocytosis to be responsible for dsRNA uptake in S. sclerotiorum. Next, I examined whether HIGS would provide effective protection. I created transgenic B. napus to target a gene involved in S. sclerotiorum pathogenicity and found significantly enhanced tolerance to infection in leaf, stem and whole plant assays. I then characterized the modes of protection by sequencing the mRNA and small RNA transcriptomes as well as performing microscopy at the site of infection. We found that the plant defense response was significantly heightened within the RNAi line, indicating that the produced dsRNA worked in tandem with innate plant defense to provide an effective defense response. Lastly, in order to gain further appreciation for this pathosystem, I studied the small RNA landscape during the B. napus – S. sclerotiorum interaction in hopes of using their innate biological mechanisms as a means to identify further gene targets for RNAi. Together, these findings provide compelling evidence and pave the way for further development of RNAi-based pathogen management strategies using both exogenous applications and transgenic approaches.
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RNA interference