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.

HCPro-mediated transmission by aphids of purified virions does not require its silencing suppression function and correlates with its ability to coat cell microtubules in loss-of-function mutant studies.

Native and amino acid (aa) substitution mutants of HCPro from potato virus Y (PVY) were transiently expressed in Nicotiana benthamiana leaves. Properties of those HCPro variants with regard to silencing suppression activities, mediation of viral transmission by aphids, and subcellular localization dynamics, were determined. One mutant failed to suppress silencing in agropatch assays, but could efficiently mediate the transmission by aphids of purified virions. This mutant also retained the ability to translocate to microtubules (MTs) in stressed cells. By contrast, another single aa substitution mutant displayed native-like silencing suppression activity in agropatch assays, but could not mediate transmission of PVY virions by aphids, and could not relocate to MTs. Our data show that silencing suppression by HCPro is not required in the aphid-mediated transmission of purified virions. In addition, since the same single aa alteration compromised both, viral transmission and coating of MTs, those two properties could be functionally related.