Abdolbaset Azizi, Luis E. del Río Mendoza
Development of RNA silencing technologies offer new options to manage plant diseases. During the silencing process the minimum free energy (MFE) of the siRNA/miRNA structures used can significantly influence their ability to silence the target genes. Therefore, this research focused on the impact of MFE in silencing of four S. sclerotiorum genes, Chitin binding domain, Mitogen-activated protein kinase, oxaloacetate acetylhydrolase, and Abhydrolase-3. For this, double-stranded RNA (dsRNA), individually and as a combination of genes, was synthesized in vitro. The hairpin RNA (hpRNA) from these genes as a tandem repeat sequence was synthesized in vitro and in vivo. Treating S. sclerotiorum mycelial colonies with 7 ng/µl of dsRNA completely silenced all target genes when compared to the control, independently of their MFE. In a separate study, the application of 0.12 ng/µl of low MFE (LMFE) hpRNA resulted in greater ( P = 0.05) silencing of all genes compared to the control and of three of the four genes when compared to the high MFE (HMFE) sequences. Spraying canola ( Brassica napus ) plants with LMFE hpRNA reduced ( P = 0.05) disease severity compared to control and to the HMFE hpRNA. Additionally, the presence of a spacer in hpRNA increased ( P = 0.05) silencing efficiency. Under the conditions of this study, the MFE and RNA spacers evaluated played a critical role in the efficacy of RNA silencing and should be considered when designing strategies for pathogen control.