title: Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: formation of dense fluorescent aggregates for sensitive virus tracking.
abstract: A series of Wheat streak mosaic virus (WSMV)-based expression vectors were developed by engineering a cycle 3 GFP (GFP) cistron between P1 and HC-Pro cistrons with several catalytic/cleavage peptides at the C-terminus of GFP. WSMV-GFP vectors with the Foot-and-mouth disease virus 1D/2A or 2A catalytic peptides cleaved GFP from HC-Pro but expressed GFP inefficiently. WSMV-GFP vectors with homologous NIa-Pro heptapeptide cleavage sites did not release GFP from HC-Pro, but efficiently expressed GFP as dense fluorescent aggregates. However, insertion of one or two spacer amino acids on either side of NIb/CP heptapeptide cleavage site or deletion in HC-Pro cistron improved processing by NIa-Pro. WSMV-GFP vectors were remarkably stable in wheat for seven serial passages and for 120 days postinoculation. Mite transmission efficiencies of WSMV-GFP vectors correlated with the amount of free GFP produced. WSMV-GFP vectors infected the same range of cereal hosts as wild-type virus, and GFP fluorescence was detected in most wheat tissues.
authors.affiliation: United States Department of Agriculture-Agricultural Research Service, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. Satyanarayana.tatineni@ars.usda.gov
authors.forename: Satyanarayana
authors.initials: S
authors.lastname: Tatineni
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: McMechan
authors.forename: Gary L
authors.initials: GL
authors.lastname: Hein
authors.forename: Roy
authors.initials: R
authors.lastname: French
mesh_terms: D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D018014:Gene Transfer Techniques; D014644:Genetic Variation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006109:Poaceae / Q000235:genetics / Q000378:metabolism* / Q000821:virology*; D019176:Potyviridae / Q000235:genetics / Q000378:metabolism*; D014908:Triticum
chemical_list: D049452:Green Fluorescent Proteins