AbstractTomato 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.