Infectious RNA transcripts were generated from full-length cDNA clones of the tobacco etch potyvirus genome containing an insertion of the bacterial I3-glucuronidase (GUS) gene between the polyprotein-coding sequences for the N-terminal 35-kDa proteinase and the helper componentproteinase. The recombinant virus was able to spread systemically in plants and accumulated to a level comparable with wild-type tobacco etch potyvirus. Proteolytic processing mediated by the 35-kDa proteinase and helper componentproteinase resulted in production of an enzymatically active GUS-helper component-proteinase fusion protein. A virus passage line that retained the GUS insert after numerous plant-to-plant transfers, as well as a line that sui a deletion of the GUS sequence, was recovered. Use of an in situ histochemical GUS assay in time-course experiments allowed the visualization of virus activity in single, mechanically inoculated leaf epidermal cells, in neighboring epidermal and mesophyll cells, in phloem-associated cells after long-ditance transport, and in cells surrounding vascular tissues of organs above and below the site of inoculation. This system represents a powerful tool to study plant virus replication, short-and long-distance virus movement, and virus-host interactions.Additionally, we show that potyviruses may serve as highly efficient, autonomously replicating vectors for the expression of foreign genes in plants.Tobacco etch virus (TEV), a well-characterized potyvirus, contains a positive-strand RNA genome of 9.5 kilobases (kb) coding for a single, large polyprotein that is processed by three virus-specific proteinases ( Fig. 1 and refs. 1 and 2). The nuclear inclusion protein "a" proteinase is involved in the maturation of several replication-associated proteins and capsid protein (3). The helper component-proteinase (HCPro) and 35-kDa proteinase both catalyze cleavage only at their respective C termini (4, 5). The proteolytic domain in each of these proteins is located near the C terminus. For HC-Pro, the N-terminal domain is required for virus transmission by aphids (6). The 35-kDa proteinase and HC-Pro derive from the N-terminal region of the TEV polyprotein (Fig. 1). Unlike the events associated with proteolytic processing, relatively little is known regarding the requirements for potyviral RNA replication and virus transport.The production of full-length, infectious RNA transcripts from cloned cDNAs representing plant positive-strand RNA virus genomes has permitted the analysis of many viral functions, including RNA replication and cell-to-cell movement (e.g., refs. 7-10). In addition, infectious transcripts from cDNA have allowed the construction of RNA-based vectors for expression of foreign genes in plants (11)(12)(13). Although a high level of replication is an attractive feature of a putative RNA virus vector, the use of RNA vectors has been limited due to instability of inserted foreign sequences and disruption of systemic virus spread after replacement of virus genes involved in movement....