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

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.