Double-stranded RNA uptake for the control of the maize pathogen Cercospora zeina

Abstract

RNA interference (RNAi) using double-stranded RNA (dsRNA) against fungal pathogens is an emerging field of crop disease control. We aimed to evaluate RNAi against the fungus Cercospora zeina causing grey leaf spot (GLS) disease on maize. Orthologues of Dicer-like 1, Dicer-like 2, RNA-dependent RNA polymerase and two copies of Argonaute were identified in the C. zeina genome and were shown to be expressed in vitro and in planta. Confocal microscopy showed that C. zeina took up exogenously applied dsRNA labelled with fluorescein. GFP-transgenic C. zeina was treated with GFP-specific dsRNA, and GFP mRNA expression and protein fluorescence were reduced by 57% and 61%, respectively. A Cz3-dsRNA targeting C. zeina chitin synthase D (CHSD), phosphatidylserine decarboxylase proenzyme 3 (PSD3) and extracellular protein 2 (ECP2) was constructed. Treatment of C. zeina cultures with the Cz3-dsRNA reduced CHSD expression by 47% and reduced cell viability by 34%. Maize leaves were inoculated with C. zeina conidia, and Cz3-dsRNA was applied either with the conidia or 16 h later. GLS disease was significantly reduced compared to the water control for the 16 h post-inoculation (hpi) treatment with Cz3-dsRNA, but not for the GFP-dsRNA specificity control or treatments at 0 hpi. We hypothesized that germination of C. zeina conidia was required for effective dsRNA-mediated control, and this was borne out by microscopy observations that most of the C. zeina conidia (70%) germinated successfully on the maize leaf surface within 16 hpi. This work lays the groundwork for a dsRNA-based fungicide against this foliar pathogen.