Investigating the off-target effects of Sclerotinia sclerotiorum double-stranded RNAs with other important fungal pathogens, Botrytis cinerea and Leptosphaeria maculans.

Abstract

Sclerotinia sclerotiorum, Botrytis cinerea, and Leptosphaeria maculans are three devastating plant fungal pathogens that affect a wide array of economically important crops. Current control strategies rely on broad spectrum fungicides; however, they are not a viable long-term solution due to the rise of fungicide resistance and their potential to harm the environment. Consequently, there is a direct and urgent need to develop new solutions to protect our crops. RNA interference (RNAi) is an intrinsic cellular mechanism that can reduce messenger RNA transcript levels through the introduction of double-stranded RNA (dsRNA). Due to the sequence specificity of RNAi, there is potential to design dsRNAs that perturb gene expression in a single pathogenic fungus without affecting other species. Using a combination of quantitative-real time polymerase chain reaction and fungal growth assays, I identified a set of novel S. sclerotiorum dsRNAs that are effective at reducing target transcript abundance and fungal growth. The potential off-target effects of these S. sclerotiorum-specific dsRNAs were subsequently examined in the closely-related B. cinerea and the more distally-related L. maculans. Data revealed that topically-applied dsRNAs can indeed be designed to be species-specific, but if even a single 21 nucleotide sequence is shared with another fungal species, cross-species transcript knockdown and reduced fungal growth can occur. This research provides useful data to evaluate the specificity of topically-applied dsRNA molecules and compelling evidence for the efficacy of RNAi-based crop protection strategies.