ABSTRACT
Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus of the order Bunyavirales, family Tospoviridae, genus Orthotospovirus. TSWV infects a broad range of plant species, causing serious economic losses. Despite its agronomic importance, molecular biological understanding of TSWV has been limited, partly due to the lack of a reverse genetics system, which would enable genetic manipulation of the virus. Here, we report that RNA synthesis by TSWV RNA polymerase occurs in the yeast Saccharomyces cerevisiae using a segment of the TSWV genome, S RNA expressed from cloned cDNA, as a template. Viral nucleocapsid protein was required for RNA synthesis. Replacement of the protein-coding and intergenic regions of TSWV S RNA by a yellow fluorescent protein (YFP)-coding sequence drastically increased the accumulation of both sense and antisense strands of the RNA, showing that this RNA was replicated. Using this system, we revealed that efficient RNA synthesis by TSWV RNA polymerase in yeast requires the 5'-terminal 17-nt and 3'-terminal ~50-nt regions of the TSWV S cRNA (complementary RNA to the genomic RNA) template.
Subject(s)
RNA/genetics , Replicon/genetics , Tospovirus/genetics , Virus Replication/genetics , Solanum lycopersicum/genetics , Solanum lycopersicum/virology , Plant Diseases/genetics , Plant Diseases/virology , RNA, Viral/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/virology , Tospovirus/pathogenicityABSTRACT
RNA-induced silencing complexes (RISCs) play central roles in posttranscriptional gene silencing. In plants, the mechanism of RISC assembly has remained elusive due to the lack of cell-free systems that recapitulate the process. In this report, we demonstrate that plant AGO1 protein synthesized by in vitro translation using an extract of evacuolated tobacco protoplasts incorporates synthetic small interfering RNA (siRNA) and microRNA (miRNA) duplexes to form RISCs that sequester the single-stranded siRNA guide strand and miRNA strand, respectively. The formed RISCs were able to recognize and cleave the complementary target RNAs. In this system, the siRNA duplex was incorporated into HSP90-bound AGO1, and subsequent removal of the passenger strand was triggered by ATP hydrolysis by HSP90. Removal of the siRNA passenger strand required the ribonuclease activity of AGO1, while that of the miRNA star strand did not. Based on these results, the mechanism of plant RISC formation is discussed.
