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

• Published in: The Plant journal : for cell and molecular biology Vol. 23; no. 4; pp. 547 - 555
• Main Authors: Choi, Il‐Ryong, Stenger, Drake C., Morris, T. Jack, French, Roy
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.08.2000; Blackwell Science
• Subjects: Agronomy. Soil science and plant productions; Avena sativa; beta-glucuronidase; Biological and medical sciences; Biotechnology; Carrier Proteins; Carrier Proteins - genetics; Carrier Proteins - metabolism; cereals; coat proteins; Edible Grain; Edible Grain - genetics; Edible Grain - metabolism; Edible Grain - virology; Fundamental and applied biological sciences. Psychology; gene expression; Gene Transfer Techniques; Genes, Reporter; Genetic engineering; Genetic engineering applications; Genetic technics; genetic transformation; Genetic Vectors; genetics; Genetics and breeding of economic plants; Glucuronidase; Glucuronidase - genetics; Glucuronidase - metabolism; GUS gene; histochemistry; Hordeum vulgare; infection; interspecific variation; leaves; metabolism; Methods. Procedures. Technologies; monocotyledonous plants; Mosaic Viruses; Mosaic Viruses - genetics; NIb protein; NPT II gene; nptII gene; open reading frames; Plant breeding: fundamental aspects and methodology; Plant Roots; Plant Roots - metabolism; polymerase chain reaction; reporter genes; Reverse Transcriptase Polymerase Chain Reaction; roots; seedlings; Sodium-Phosphate Cotransporter Proteins; Symporters; Transgenic animals and transgenic plants; Transgenic plants; Triticum aestivum; virology; virulence; virus gene expression vector; Wheat streak mosaic virus; Zea mays; Monocotyledones; β-Glucuronidase; Genetic transformation; Zea mays; Plant pathogen; Enzyme; Plant juvenile growth stage; Insertion; Gene expression; Cereal crop; Virus; Gramineae; Glycosidases; Angiospermae; Cereal; Hydrolases; Spermatophyta; Genetic engineering; Transgenic plant; O-Glycosidases; Vector; Triticum aestivum
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1046/j.1365-313x.2000.00820.x

Summary Inserts bearing the coding sequences of NPT II and β‐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, permitting excision of foreign protein domains from the viral polyprotein. Wheat, barley, oat and maize seedlings supported systemic infection of WSMV bearing NPT II. The NPT II insert was stable for at least 18–30 days post‐inoculation and had little effect on WSMV CP accumulation. Histochemical assays indicated the presence of functional GUS protein in systemically infected wheat and barley plants inoculated with WSMV bearing GUS. The GUS constructs had greatly reduced virulence on both oat and maize. RT–PCR indicated that the GUS insert was subject to deletion, particularly when expressed as a GUS–NIb protein fusion. Both reporter genes were expressed in wheat roots at levels comparable to those observed in leaves. These results clearly demonstrate the utility of WSMV as a transient gene expression 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.