siRNA-Mediated Silencing of Antifungal Resistance Genes: A Research Protocol

Abstract

Introduction: Antifungal resistance (AFR) is an underrepresented issue that threatens both global health and food security. A common feature of many pathogenic fungi is their ability to produce RNA-induced silencing complexes (RISC) to protect against mycoviruses, thereby silencing the expression of targeted genes. Herein, we aim to create a genetically-modified mycovirus which can silence AFR genes specific to tebuconazole by leveraging the RISC silencing mechanism against the fungi’s native genes.

Methods: To investigate the possible effects of mycoviruses on AFR, Fusarium graminearum (Fg) cultures will be infected with modified Fusarium graminearum deltaflexivirus 1 (mFgDFV1), each of which contain a 600 nt Fg ATP-binding cassette 3 (FgABC3) segment (an azole resistance gene). mFgDFV1 will be produced from Saccharomyces cerevisiae via an episomal plasmid and subsequently purified using an aqueous two-phase system. Thereafter, a Western and Northern blot will be employed to confirm successful mFgDFV1 synthesis. The efficacy of mFgDFV1 on repressing AFR will be evaluated by comparing the minimum inhibitory concentration (MIC₅₀ and MIC₉₀) of tebuconazole for Fg groups treated with mFgDFV1, wild-type FgDFV1, or no virus via protoplast fusion.

Results: Upon completion of the experiments above, 3 sets of MIC₅₀ and MIC₉₀ values will be obtained. Each set will correspond to either mFgDFV1 treatment, wild-type FgDFV1 control, or water control. It is expected that Fg treated with mFgDFV1 will induce RISC, silencing FgABC3 and thus lowering MIC₅₀ and MIC₉₀ relative to both controls.

Discussion: If effective, this approach to addressing AFR could be advantageous given the utility of RISC in fungi (e.g., if fungi downregulate the RISC response, they would become more susceptible to other viruses). Moreover, this method could be translated to a variety of other genetic and fungal targets if desired.

Conclusion: This article presents a method to effectively overcome antifungal resistance by exploiting the fungal short interfering RNA defense mechanism. Should this experiment be successful, this modified Fg virus treatment could potentially stop multidrug-resistant Fg infestations, although further experimentation is required. Future studies could study the effectiveness of other antifungal resistant fungi and/or examine the biosafety and ecological footprint of this method.