Optimization of in vitro RNA interference assays to reduce variability in target gene knockdown in the phytopathogen Sclerotinia sclerotiorum
| dc.contributor.author | Fatema, Reemana | |
| dc.contributor.examiningcommittee | Gerstein, Aleeza (Microbiology) | |
| dc.contributor.examiningcommittee | Marcus, Jeffrey (Biological Sciences) | |
| dc.contributor.supervisor | Whyard, Steve | |
| dc.date.accessioned | 2025-02-18T15:45:06Z | |
| dc.date.available | 2025-02-18T15:45:06Z | |
| dc.date.issued | 2025-01-16 | |
| dc.date.submitted | 2025-01-16T21:48:12Z | en_US |
| dc.degree.discipline | Biological Sciences | |
| dc.degree.level | Master of Science (M.Sc.) | |
| dc.description.abstract | Sclerotinia sclerotiorum is a broad host range fungal phytopathogen that is the causal agent of Sclerotinia stem rot in many crop plants, causing devastating yield losses worldwide. Sclerotinia is partially controlled using broad-spectrum chemical fungicides, but new, more species-specific fungicides, such as double-stranded RNAs, are being considered as safer alternatives. This study evaluated the effectiveness of long double-stranded RNAs (dsRNAs) and paperclip RNAs (pcRNAs) to induce gene silencing in two different fungal inoculants of S. sclerotiorum. The three different genes that were targeted in this study, SS1G_01703, SS1G_00005, SS1G_14116, are all involved in different aspects of S. sclerotiorum’s infection process i.e. pathogenicity, encoding 60S ribosomal unit and mycelial growth respectively. Initial dsRNA experiments using small fungal plugs collected from solid media generally resulted in highly variable levels of transcript knockdown, potentially arising from unequal hyphal amounts in treatment tubes. This led to the development of fungal slurries as the inoculum to provide a more uniform distribution of hyphae across all the samples. The slurry method resulted in significant transcript knockdowns using all three long dsRNAs at different doses, although some variability persisted. The variability using the slurry method was linked to potential differences in hyphal developmental stages, age of hyphae and differences in expression levels of each target gene. In contrast, structurally different and shorter in length pcRNAs failed to induce consistent transcript knockdown in both plug and slurry assays, despite earlier reports of their efficacy in reducing lesion sizes on canola leaves. This inefficacy may stem from pcRNAs' reliance on a single siRNA for gene targeting, potentially insufficient to overcome the structural complexity of target mRNAs. These findings highlighted the potential of long dsRNAs for gene knockdown in S. sclerotiorum using optimized slurry-based assays, while suggesting that pcRNAs require further modifications. Future work can explore alternative siRNA designs, increased pcRNA doses, and identifying accessible mRNA regions to improve knockdown efficiency. Additionally, optimizing the methods to assess hyphal developmental stages and minimizing variability during sample preparation are crucial for reproducibility and accuracy in RNAi-based studies. | |
| dc.description.note | May 2025 | |
| dc.identifier.uri | http://hdl.handle.net/1993/38891 | |
| dc.language.iso | eng | |
| dc.subject | RNA interference | |
| dc.subject | Double-stranded RNA | |
| dc.subject | siRNA | |
| dc.subject | Sclerotinia | |
| dc.title | Optimization of in vitro RNA interference assays to reduce variability in target gene knockdown in the phytopathogen Sclerotinia sclerotiorum | |
| local.subject.manitoba | yes | |
| oaire.awardTitle | University of Manitoba Graduate Fellowship (UMGF) |