Differences in Virulence among PVY Isolates of Different Geographical Origins When Infecting an Experimental Host under Two Growing Environments Are Not Determined by HCPro.

The contribution of the HCPro factors expressed by several PVY isolates of different geographical origins (one from Scotland, one from Spain, and several from Tunisia) to differences in their virulence in Nicotiana benthamiana plants was investigated under two growing conditions: standard (st; 26 °C and current ambient levels of CO2), and climate change-associated (cc; 31 °C and elevated levels of CO2). In all cases, relative infection symptoms and viral titers were determined. The viral HCPro cistrons were also sequenced and amino-acid features of the encoded proteins were established, as well as phylogenetic distances. Additionally, the abilities of the HCPros of several isolates to suppress silencing were assessed under either growing condition. Overall, viral titers and infection symptoms decreased under cc vs. st conditions. However, within each growing condition, relative titers and symptoms were found to be isolate-specific, with titers and symptom severities not always correlating. Crucially, isolates expressing identical HCPros displayed different symptoms. In addition, all HCPro variants tested displayed comparable silencing suppression strengths. Therefore, HCPro alone could not be the main determinant of the relative differences in pathogenicity observed among the PVY isolates tested in this host, under the environments considered.

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.

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 Escherichia coli-encapsulated dsRNA compared to naked dsRNA against single and dual infection by Potato virus X expressing the green fluorescent protein (PVX-GFP) and Potato virus Y (PVY) in Nicotiana benthamiana. We found that, in our conditions, the effectiveness of E. coli-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 N. benthamiana. 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.