Abstract
Plant RNA viruses are obligate intracellular parasites that hijack specific cellular membranes to replicate their genomes in what are commonly known as viral replication complexes (VRC). These contain host- and virus-encoded proteins and viral RNA. Double-stranded RNA (dsRNA) is a mandatory intermediate of RNA replication and a hallmark feature of VRCs. We have recently developed a method to isolate viral dsRNA and its associated proteins through pull-down of an ectopically expressed dsRNA-binding protein (B2:GFP) from infected Arabidopsis thaliana plants. After mass spectrometry analysis to identify the dsRNA-associated proteins, resulting candidate proteins of interest are tagged with a red fluorescent protein and their subcellular localization in relation to VRCs is assessed by transient expression within leaves of B2:GFP-transgenic Nicotiana benthamiana plants. In this chapter we describe in detail these experimental procedures to allow investigators to characterize the replication complexes of their plant RNA virus of interest.
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