Tagging of plant potyvirus replication and movement by insertion of beta-glucuronidase into the viral polyprotein.

Abstract

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....

Long-distance movement of cherry leaf roll virus in infected tobacco plants

“…Very clear hybridization signals were observed within the major vein of the leaves, indicating that the virus probably replicates in vascular tissue. This is contrary to observations by Leisner et al (1992) in turnip plants infected with CaMV and by Dolja et al (1992) in tobacco plants infected with TEV where virus accumulation was significantly less in major than in minor veins and mesophyll cells. The foci of hybridization resulting from a non-vascular symptomatic area were only present when the lateral vein situated immediately downward had a viral signal, indicating that minor veins are the most frequent pathways for invasion of parenchyma (note that in the right part of leaf 3 in Fig.…”

Cloning of the Arabidopsis RTM1 gene, which controls restriction of long-distance movement of tobacco etch virus

“…Transformants were transferred to soil and grown for 15-20 days before inoculation. Rosette leaves were dusted with carborundum and inoculated with TEV-GUS, a recombinant TEV strain encoding GUS (17). GUS activity assays were done by using inflorescence tissue at 15 and 20 days postinoculation (11).…”

“…GUS activity assays were done by using inflorescence tissue at 15 and 20 days postinoculation (11). Inoculated leaves from plants that scored negative in GUS activity assays of systemic tissue were tested for infection by in situ histochemical assay (17).…”

A hypersensitive response-like mechanism is involved in resistance of potato plants bearing the Ry(sto) gene to the potyviruses potato virus Y and tobacco etch virus.

“…In previous experiments (Hinrichs et al, 1997) we were able to infect potato cultivars which were susceptible to PVY with TEV, but we were unable to generate systemic infections in cultivars carrying the Ry sto gene. Because the same type of necrotic reaction was produced by PVY and TEV in the same cultivars and because resistance based on Ry sto or a closely linked gene seemed to be effective against both viruses, we decided to study the resistance reaction in potato to TEV in detail with the aid of a TEV-GUS construct (Dolja et al, 1992). TEV-GUS has the advantage that it carries the Escherichia coli uidA gene (GUS) as a reporter gene to monitor the spread of virus in the plant.…”

“…In situ GUS assays were performed by vacuum infiltration of potato leaves with the colorimetric substrate X-gluc (5-bromo-4-chloro-3-indolyl β--glucuronic acid, cyclohexylammonium salt ; from GBT, St Louis, Mo., USA) according to Jefferson (1987) as modified by Dolja et al (1992). Infiltrated leaves were incubated overnight at 37 mC before they were cleared with 70 % ethanol.…”