ANTI-SILENCING · Molecular mechanisms of silencing suppression in plants

Final Activity Report Summary - ANTI-SILENCING (Molecular mechanisms of silencing suppression in plants)

Small RNAs and their role in posttranscriptional silencing is the latest fascinating paradigm in molecular biology. In response to viral invasion an 'adaptive immune-system' is induced based on the recognition of double-stranded (ds)RNA which is produced during virus infection. dsRNA is recognised by 'Dicer', an RNAse III-like enzyme and 'diced' into classes of 'small interfering RNAs', 21 to 25 nucleotides in length, that get stabilised by Hen1-mediated 3'-end methylation. These original double-stranded fragments are unwound by a helicase and one of the single strands builds an 'RNA-induced silencing complex' (RISC). RISC and related complexes have various functions. Firstly, they interact with and cleave target RNA. Secondly, they can interact with target RNAs and produce new dsRNA with the siRNA as a primer and RNA dependent RNA polymerase (RdRp) as an enzyme. Thirdly, in the nucleus they can scan the chromatin for cognate DNA and modify it by RNA dependent DNA methylation. Apparently, silencing in plants is so successful, that plant viruses cannot infect a plant without a counter defence mechanism. Depending on the virus, silencing suppression can be a secondary function of almost any type of viral protein (coat protein, protease, movement protein, etc.) and act on various steps of the silencing pathway. The information about the molecular mechanisms of silencing suppression remains limited. In order to clarify which step of silencing pathway is affected by suppressors we set up in vitro systems to detect Dicer, RISC, RdRp and Hen1 activities. Further, we cloned, expressed and purified several known and presumable silencing suppressors from various viruses, namely: tombusvirus P19, polerovirus P0, carmovirus TCV coat protein (CP), carlovirus PVM ORF6 and geminivirus (Cotton leaf curl begomovirus) betaC1, C4 and V2 proteins. We checked ability of these proteins to inhibit separate steps of silencing pathway. One of the mechanisms of silencing suppression is direct binding of suppressors to long and short dsRNA. We installed method to measure RNA-binding ability of suppressor protein and determined the efficiency of RNA-binding of these proteins. We studied molecular mechanism of silencing suppression by tobamovirus TMV. We demonstrated that short fragment of polymerase p126 also known as TMV methyltransferase have suppression function and prevents methylation of siRNA by Hen1 protein. In addition, we studied plant Dicer complexes. Using biochemical methods we estimated molecular weights of two Dicer complexes, ionic and energy requirements, temperature and pH optimums, substrate specificity.

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