Diverse and Newly Recognized Effects Associated with Short Interfering RNA Binding Site Modifications on the Tomato Bushy Stunt Virus P19 Silencing Suppressor

The Tomato bushy stunt virus-encoded P19 forms dimers that bind duplex short interfering RNAs (siRNAs) to suppress RNA silencing. P19 is also involved in multiple host-specific activities, including the elicitation of symptoms, and in local and/or systemic spread. To study the correlation between those various roles and the siRNA binding by P19, predicted siRNA-interacting sites were modified. Twenty-two mutants were generated and inoculated onto Nicotiana benthamiana plants, to reveal that (i) they were all infectious, (ii) symptom differences did not correlate strictly with mutation-associated variation in P19 accumulation, and (iii) substitutions affecting a central domain of P19 generally exhibited symptoms more severe than for mutations affecting peripheral regions. Three mutants selected to represent separate phenotypic categories all displayed a substantially reduced ability to sequester siRNA. Consequently, these three mutants were compromised for systemic virus spread in P19-dependent hosts but had differential plant species-dependent effects on the symptom severity. One mutant in particular caused relatively exacerbated symptoms, exemplified by extensive morphological leaf deformations in N. benthamiana; this was especially remarkable because P19 was undetectable. Another striking feature of this mutant was that only within a few days after infection, viral RNA was cleared by silencing. One more original property was that host RNAs and proteins (notably, the P19-interactive Hin19 protein) were also susceptible to degradation in these infected N. benthamiana plants but not in spinach. In conclusion, even though siRNA binding by P19 is a key functional property, compromised siRNA sequestration can result in novel and diverse host-dependent properties.