Graduate Thesis Or Dissertation
 

Host-Induced Gene Silencing in Black Cottonwood for Control of Septoria Canker: Efficacy and Non-Target Impacts

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  • Septoria canker remains the most important disease of poplars in intensively managed forest plantations. Genetic resistance has long been considered the best way to manage for this disease. Transgenic resistance mediated by RNA silencing against pathogens and pests (HIGS: host-induced gene silencing) has shown promise in other pathosystems but has never been tested in a forest tree against a fungal pathogen. The effects of HIGS are expected to be specific to the target organism and closely related taxa; however, this expectation has never been evaluated empirically against fungal pathogens under field conditions. In addition, the effects of plant transformation and associated in vitro propagation and sterile culture on subsequent microbial community assembly have never been previously evaluated, but may be an important unintended effect of HIGS technology. To answer these questions, we studied the direct effects of synthetic double-stranded RNAs (dsRNAs) on the target pathogen in axenic culture, we created transgenic poplars that contained HIGS transgenes, and we evaluated their disease resistance in the greenhouse, and their foliar fungal community composition in the laboratory and the field. Our first study explored HIGS as a novel means to engineer resistance to Septoria canker caused by the fungal pathogen Sphaerulina musiva. HIGS transgenic poplars expressing double-stranded RNA (dsRNA) that targeted either or both S. musiva CYP51 (P450 lanosterol C14α-demethylase) and DCL (dicer-like) genes were screened for resistance to stem canker disease in two greenhouse inoculation trials. We did not detect statistically significant differences in disease severity between transgenic lines and wild-type controls. The correlation between variation in greenhouse-expressed disease severity and transgene expression was statistically significant for HIGS events targeting DCL but not for HIGS events targeting CYP51. In vitro studies with synthetic dsRNAs tested: (1) S. musiva’s capacity for uptake of environmental dsRNA; (2) effects of in vitro silencing of CYP51 and DCL on fungal growth and target transcript abundance; and 3) persistence of dsRNA in culture. We were unable to detect uptake of fluorescently tagged dsRNA in vitro with confocal live imaging, though using the same methods we were able to detect uptake in another fungal pathogen known to be amenable to HIGS, suggesting a very limited capacity for dsRNA uptake by S. musiva. In dsRNA-treated cultures, we were unable to detect fungal growth inhibition, and RNA was rapidly degraded in the culture medium. Of five target transcripts tested after dsRNA treatment, only one had significantly impaired expression. These results, together with our disease screening, suggest that HIGS and the related technique SIGS (spray-induced gene silencing with dsRNAs) are unlikely to be effective control measures for this disease. In our second study we used ITS metabarcoding to evaluate the effects of HIGS transgenes, and the effects of plant transformation—including micropropagation, antibiotic selection, and organogenesis—on the foliar fungal microbiome of field-grown trees over two seasons. Micropropagation-derived trees grown in a greenhouse were largely uncolonized after 50 days. Once established in the field, after a single growing season, communities contained 372 operational taxonomic units (OTUs) and samples were as rich as those of trees of the same genotype that had been growing at a nearby location for several years. We did not detect any effects from micropropagation, or from the use of antibiotic selection and organogenesis during Agrobacterium transformation, on subsequent leaf fungal communities. The expression of HIGS constructs directed against the Septoria canker fungus also had no detectable effect on any non-target fungal taxa, including the very closely related species Sphaerulina populicola. Our results suggest that micropropagation, genetic transformation and associated antibiotic selection and organogenesis, and the expression of antifungal HIGS genes, had minimal, if any, impacts on subsequent foliar microbiomes of field-grown trees.
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