Silencing Arbuscular Mycorrhizal Fungal Gene Using Chitosan Nanoparticle-Mediated dsRNA Delivery System

It has been discovered that many phytopathogenic fungi can absorb exogenous double-stranded RNAs (dsRNAs) to silence target genes, inhibiting fungal growth and pathogenicity for plant protection. In our recent report, the beneficial arbuscular mycorrhizal (AM) fungi are capable of acquiring external naked dsRNAs; however, whether the dsRNAs can be delivered into AM fungi through nanocarriers remains to be investigated. Here, we introduce a simple and advanced method for in vitro synthesizing chitosan (CS)/dsRNA polyplex nanoparticles (PNs) to silence the target gene in the AM fungus <i>Rhizophagus irregularis</i>. This method is straightforward, requiring minimal modifications, and is both efficient and eco-friendly, offering potential for rapid application in elucidating gene functions in AM fungi. Key features • The chitosan can carry the dsRNA derived from the AM fungus <i>Rhizophagus irregularis</i>. • CS/dsRNA polyplex nanoparticles (PNs) can successfully silence the target gene in the AM fungus <i>R. irregularis</i>. • CS/dsRNA PNs can be applied to the characterization of AM fungal genes via the spray-induced gene silencing (SIGS) approach. • This protocol can be applied in asymbiotic and symbiotic cultures of AM fungi. Graphical overview Overview of the chitosan/dsRNA gene silencing procedures.