Double-Stranded RNAs Targeting <i>Sphaerulina musiva</i> Silence <i>DCL</i> but Not <i>CYP51</i> In Vitro and Fail to Confer Resistance to Canker in Transgenic Poplar

Host-induced gene silencing (HIGS) is a common method for engineering plant protection against pathogens, although success requires double-stranded RNA (dsRNA) uptake mechanisms that may not be present in all fungi. We explored HIGS in transgenic poplar to study and control Sphaerulina musiva, the cause of Septoria stem canker disease. HIGS transgenic poplars expressing dsRNA that targeted either or both S. musiva CYP51 and DCL were developed and screened for resistance to stem canker disease in two greenhouse inoculation trials. While differences in resistance between transgenic lines and wild-type controls were not detected, there was a correlation between greenhouse-expressed disease resistance and transgene expression among HIGS lines targeting S. musiva DCL. To evaluate the likelihood that HIGS or spray-induced gene silencing might be effective under some conditions, concurrent with greenhouse screening, we studied: (i) S. musiva’s capacity for uptake of environmental dsRNA; (ii) effects of in vitro silencing of CYP51 and DCL on fungal growth and target transcript abundance; and (iii) persistence of dsRNA in culture. The uptake of fluorescently tagged dsRNA was not detected with confocal imaging. In dsRNA-treated cultures, fungal growth inhibition was not detected, and RNA was rapidly degraded. Of the five target transcripts tested after dsRNA treatment, only DCL1 had reduced expression. Knockdown of DCL1 along with the enhanced resistance among high-expressing HIGS events targeting DCL suggests some HIGS may have been observed. Further determination of the factors limiting dsRNA uptake by S. musiva are needed to determine whether HIGS can be an effective technology for limiting stem canker. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .