Viral replication and antiviral immune response in a plant in vitro system

By applying cytoplasmic extracts of Nicotiana tabacum BY2 protoplasts (‘BYL’) we were able to establish an experimentalsystem that reconstitutes the replication of the (+)RNA plant virus Tomato Bushy Stunt Virus (TBSV) in vitro. After synthesis of the TBSV proteins p33 and p92 in the BYL by in vitro translation, viral replication complexes (RC) were formed that catalyze the synthesis of progeny viral (+)RNAs via (-) RNA intermediates. The TBSV RC contains various cellular ‘host’ proteins that are proposed to support viral replication but are as yet incompletely characterized. In our project, we applied the BYL to specifically identify TBSV RNA-binding host factors. Two RNA helicases were recognized that significantly stimulate viral RNA replication. Hence, one aim of the current application will be to understand the function(s) of these and other RNA helicases in TBSV replication. Further studies with the BYL in vitro system revealed that it also reconstitutes the major antiviral immune response of the plant cell; i.e., cleavage of viral RNA into vsiRNAs (viral small interfering RNAs), siRNA-programmed formation of RNA-induced silencing complexes (RISC) and blockage of TBSV RNA replication by endonucleolytic RNA cleavage. The second main aim of the application involves the identification of particularly effective vsiRNAs and to determine their activity in concert with the plant ARGONAUTE (AGO) proteins. As functional RISC can be isolated from the BYL and was found to contain several additional proteins besides AGO, we further aim to identify these factors and to understand their role in the antiviral immune response.