A plant virus vector for systemic expression of foreign genes in cereals

Inserts bearing the coding sequences of NPTII and beta-glucuronidase (GUS) were placed between the nuclear inclusion b (Nlb) and coat protein (CP) dom ains of the wheat streak mosaic virus (WSMV) polyprotein ORF. The WSMV Nlb- CP junction containing the nuclear inclusion a (Nla) protease cleavage site was duplicated, permitting excision of foreign protein domains from the vi ral polyprotein. Wheat, barley, oat and maize seedlings supported systemic infection of WSMV bearing NPTII. The NPTII insert was stable for at least 1 8-30 days post-inoculation and had little effect on WSMV CP accumulation. H istochemical assays indicated the presence of functional GUS protein in sys temically infected wheat and barley plants inoculated with WSMV bearing GUS . The GUS constructs had greatly reduced virulence on both oat and maize. R T-PCR indicated that the GUS insert was subject to deletion, particularly w hen expressed as a GUS-Nlb protein fusion. Both reporter genes were express ed in wheat roots at levels comparable to those observed in leaves. These r esults clearly demonstrate the utility of WSMV as a transient gene expressi on vector for grass species, including two important grain crops, wheat and maize. The results further indicate that both host species and the nature of inserted sequences affect the stability and expression of foreign genes delivered by engineered virus genomes.