Topical Application of Escherichia coli-Encapsulated dsRNA Induces Resistance in Nicotiana benthamiana to Potato Viruses and Involves RDR6 and Combined Activities of DCL2 and DCL4

Abstract

Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants represents an attractive alternative to transgene-based silencing approaches. However, improvement of dsRNA stability in natural conditions is required in order to provide long-term protection against the targeted virus. Here, we tested the protective effect of topical application of <i>Escherichia coli</i>-encapsulated dsRNA compared to naked dsRNA against single and dual infection by <i>Potato virus X</i> expressing the green fluorescent protein (PVX-GFP) and Potato virus Y (PVY) in <i>Nicotiana benthamiana</i>. We found that, in our conditions, the effectiveness of <i>E. coli</i>-encapsulated dsRNA in providing RNAi-mediated protection did not differ from that of naked dsRNA. dsRNA vaccination was partly effective against a dual infection by PVX-GFP and PVY, manifested by a delay in the expression of the synergistic symptoms at early times after inoculation. Using PVX-GFP as a reporter virus together with a suite of RNAi knockdown transgenic lines, we have also shown that RNA-directed RNA polymerase 6 and the combined activities of DICER-like 2 (DCL2) and DCL4 act to promote efficient resistance to virus infection conferred by topical application of dsRNA in <i>N. benthamiana</i>. Our results provide evidence that exogenous dsRNA molecules are processed by the RNA silencing pathways commonly used by the host in response to virus infection.