Tospovirus : induction and suppression of RNA silencing

While infecting their hosts, viruses must deal with host immunity. In plants the antiviral RNA silencing pathway is an important part of plant innate immunity. Tospoviruses are segmented negative-stranded RNA viruses of plants. To counteract the antiviral RNA silencing response in plants, tospoviruses have evolved a silencing suppressor function via its NSs protein. This viral protein has previously been shown to bind dsRNA that likely arises from secondary RNA folding structures in viral RNAs. The aim of the present research was to further investigate the interaction between tospoviruses and the plant antiviral RNA silencing response, including the target sequences in the viral RNA and the further role of the NSs protein as part of the tospovirus counterdefence strategy. In order to identify the target and inducer for RNA silencing against tospoviruses, small RNAs purified from plants infected with three tospoviral species, tomato spotted wilt virus (TSWV), groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), were probed against the viral RNA segments of these three different tospoviruses (Chapter 3). Virus-derived siRNAs (vsiRNAs) were found to be derived from all three genomic RNA segments but predominantly the ambisense M and S RNAs. Further profiling on the S RNA sequence revealed that vsiRNAs were found from almost the entire S RNA sequence, except the predicted AU-rich hairpin (HP) structure encoded by the intergenic region (IGR) from where hardly any vsiRNAs were found. Similar profiles were observed with the closely related GRSV as well as the distantly related TYRV. Dicer cleavage assays using Drosophila melanogaster embryo extracts showed that synthetic transcripts of the IGR-HP region were recognized as substrate for Dicer. Transient agroinfiltration assays of a GFP-sensor construct containing the IGR-HP sequence at its 3′-UTR did not show more rapid/strong silencing, and profiling of the corresponding siRNAs generated outside the context of a viral infection still revealed relatively low levels of IGR-HP-derived siRNAs. These data support the idea that the IGR-HP region/structure is a weak inducer of RNA silencing and plays a minor role in the amplification of a strong antiviral RNA silencing response. Next, a biochemical analysis was performed using E. coli-expressed and purified NSs from GRSV and TYRV. The binding of both purified NSs proteins to small and long dsRNA indicated that this is likely a generic feature of all tospoviral NSs proteins (Chapter 4). Binding of siRNAs to NSs furthermore revealed two shifts on polyacrylamide gels i.e. a first shift at low NSs concentrations followed by a second larger one at higher concentrations. When the NSs protein of TSWV resistant breaker (RB) isolates (of Tsw-gene based resistance), which lack RSS activity when transiently expressed, were analyzed using extracts from infected plants still a major (second) shift of siRNAs was observed, similar to the case with extracts containing TSWV resistant inducer (RI) isolates. In contrast, plant extracts containing transiently expressed NSs proteins alone (no infection) showed only the smaller, first shift for NSsRI but no shift for NSsRB. The ability of NSs to suppress systemic silencing is demonstrated for the NSs proteins of TSWV, GRSV and TYRV, and their relative strengths to suppress local and systemic silencing were compared (Chapter 5). A system was developed to quantify suppression via GFP silencing constructs, allowing comparison of relative RNA silencing suppressor strength. In this case NSs proteins of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSsTYRV was just as strong as those by NSsTSWV and NSsGRSV, even though NSsTYRV was expressed in lower amounts. Moreover, a set of selected NSsTSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream of the biogenesis of siRNAs and their sequestration. In summary, evidence is presented showing that sequences from all three genomic segments from tospovirus are targeted by the plant RNA silencing machinery. The predicted hairpin sequence in the IGR is poorly targeted. Biochemical experiments with purified NSs proteins further support the view that binding to small and long dsRNA is a characteristic common to all tospovirus NSs proteins. Furthermore, tospovirus NSs proteins suppress systemic silencing and there are indications that local and systemic silencing suppression can be uncoupled in NSs. Collectively, these results add to our current understanding of the tospovirus-plant interaction involving antiviral RNA silencing and the viral counter-defence (NSs protein). Lastly, the results of the research presented in this thesis are discussed in light of the current knowledge on RNA silencing and to present some future perspectives and questions that remain open and/or resulted from this thesis (Chapter 6).