Small interference RNA (siRNA)-mediated down-regulation of a cAMP-dependent protein kinase regulatory subunit (CRP) results in reduced pathogenicity in Fusarium graminearum and Magnaporthe oryzae

Abstract

The cyclic AMP (cAMP) signaling pathway is a major signal transduction pathway with multiple roles in most filamentous fungi. Utilizing RNA interference (RNAi) approach, which involves double-stranded (dsRNA) or small interference RNAs (siRNAs) for targeted gene silencing, an attempt was made to test whether CRP (cAMP protein kinase regulatory protein), a key cAMP pathway gene in fungal development and virulence, is also required for pathogenesis of F. graminearum. By supplementing axenic fungal conidia (in-vitro) as well as spray-induced gene silencing (SIGS) in the leaves of wheat and Arabidopsis (in-planta), the targeted silencing of CRP gene was performed. Our findings revealed statistically significant reduction in colony growth and fungal biomass in CRP-siRNA treated-F. graminearum at 5 and 7 days post treatment. The CRP-siRNA treatment of F. graminearum conidia at 35 nM concentration resulted in 50% growth inhibition showing abnormal conidia morphology with visible conglobate structures at hyphal tips and poor conidial germination in in-vitro studies. To validate the findings, another hemibiotrophic fungal pathogen, Magnaporthe oryzae was also treated with CRP-siRNAs in culture (in-vitro) as well as by SIGS approach on rice leaves (in-planta) prior to challenge with the pathogen. Similar results were obtained in both the pathosystems, confirming the conservancy of CRP gene across different fungal species along with its role in fungal virulence. Real time-quantitative (RT-q) PCRs showed significant down-regulation of CRP gene in both in-vitro and in-planta studies compared to the untreated control or unrelated siRNA treatment in both the pathogens, indicating the CRP gene's involvement in fungal pathogenicity.