Spray‐induced gene silencing of three G‐protein signaling genes from the arbuscular mycorrhizal fungus Rhizophagus irregularis inhibits spore germination and hyphopodium formation

About 70% of land plants form symbioses with arbuscular mycorrhizal (AM) fungi. Some plant genes important for accommodating AM fungi within roots have been characterized, but AM fungal genes involved in asymbiotic growth and hyphopodium formation remain elusive due to a lack of methods for genetic manipulation. Here, we introduce an innovative gene silencing technology based on spraying double-stranded RNA (dsRNA) to characterize the functions of three genes encoding G-protein signaling proteins, including the regulator of G-protein signaling RiRgs3, the Gα subunit RiGpa3, and the Gβ subunit RiGpb1 from the AM fungus Rhizophagus irregularis at the asymbiotic and initial symbiotic stages. RiRgs3, RiGpa3, and RiGpb1 expression is induced in the early stages of AM symbiosis. Using spray-induced gene silencing (SIGS), we discovered that R. irregularis can take up dsRNA. Moreover, SIGS of RiRgs3, RiGpa3, or RiGpb1 led to defects in spore germination and hyphopodium formation. In conclusion, our results reveal that SIGS is a suitable technique for the analysis of gene function in AM fungi and that G-protein signaling is required for spore germination and hyphopodium formation.