|Choi, Il-Ryong - UNI OF NE, LINCOLN|
|Morris, T - UNI OF NE, LINCOLN|
Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 24, 2000
Publication Date: August 1, 2000
Citation: Choi, I., Stenger, D.C., Morris, T.J., French, R.C. 2000. A plant virus vector for systemic expression of foreign genes in cereals. Plant Journal. Plant Journal 23:547-555. Interpretive Summary: Wheat streak mosaic virus (WSMV) has been modified to express foreign proteins in wheat and other cereals. The WSMV gene vector bearing the NPT II gene systemically expressed NPT II protein in mechanically inoculated wheat, barley, corn, and oat seedlings. The level of NPT II protein expressed was equivalent to that commonly obtained in transgenic plant lines. The WSMV gene vector bearing the GUS gene systemically expressed functional GUS protein in both wheat and barley. The WSMV gene vector represents the first engineered virus capable of systemically expressing foreign genes in wheat and other cereals and provides a simple means of evaluating the effects of foreign gene expression in cereals without the need for transformation and regeneration.
Technical Abstract: Inserts bearing the coding sequences of NPT II and á-glucuronidase (GUS) were placed between the nuclear inclusion b (NIb) and coat protein (CP) domains of the wheat streak mosaic virus (WSMV) polyprotein ORF. The WSMV NIb-CP junction containing the nuclear inclusion a (NIa) protease cleavage site was duplicated such that foreign protein domains were excised from the eviral polyprotein. Wheat, barley, oat, and corn seedlings supported systemic infection of WSMV bearing NPT II. NPT II protein in wheat was detected by immunoblotting, and quantitative estimates of NPT II protein accumulation in all four cereal species was accomplished by ELISA. The NPT II insert was stable for at least 18 days post inoculation and had minimal effect on the accumulation of WSMV CP. Histochemical assays indicated the presence of functional GUS protein in systemically infected wheat and barley plants inoculated with WSMV bearing GUS. RT-PCR indicated that the GUS insert was subject to deletion, particularly when the NIa protease cleavage site was not duplicated such that GUS remained fused to the NIb protein. Accumulation of CP in wheat plants inoculated with WSMV containing GUS gene inserts was delayed and reduced relative to wild type, and these effects were more pronounced when GUS was produced as a fusion protein. GUS activity in oat was restricted to clusters of cells at infection foci 3 days post inoculation, whereas in corn GUS activity was not observed at any time post inoculation. These results demonstrate utility of a plant RNA virus as a gene vector capable of systemic expression in cereals, and further indicate that both host species and inserted sequences affect the stability and expression of foreign genes delivered to cereals by an engineered virus genome.