|Mcmechan, A. Justin -|
|Hein, Gary -|
Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 30, 2010
Publication Date: January 4, 2011
Repository URL: http://hdl.handle.net/10113/49414
Citation: Tatineni, S., Mcmechan, A., Hein, G., French, R.C. 2011. 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. Virology. 410:268-281, doi:10.1016/j.virol.2010.10.043. Interpretive Summary: In this study, we inserted a GFP cistron into the WSMV genome with a range of cleavage peptides to release GFP from the viral polyprotein. The cleavage peptides from WSMV failed to release GFP from the viral polyprotein and formed a fusion protein of GFP:HC-Pro, which was expressed as dense aggregate-like fluorescent structures for sensitive tracking of the virus in cereal hosts. The availability of GFP-tagged WSMV greatly improved the ability to non-destructively trace the spread of the virus within plant tissues during the infection, and opened new research avenues examining the WSMV movement, trafficking, and virus-host interactions in cereal hosts. WSMV-GFP is one of the few viruses developed to express the GFP systemically in monocot plants. WSMV-GFP vectors were highly stable in wheat for more than 120 days after inoculation, which was close to the life span of wheat. Distribution of WSMV in wheat was examined using the GFP-tagged virus and it was found that WSMV was detected in most parts of the wheat except in the seed endosperm. WSMV-GFP vectors were transmitted by wheat curl mites, which would allow examining virus-mite interactions and the mechanism of mite transmission of WSMV. Additionally, the WSMV-GFP vectors provide valuable tools for screening the wheat cultivars for WSMV resistance.
Technical Abstract: A series of Wheat streak mosaic virus (WSMV)-based expression vectors were developed by engineering 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 recognition heptapeptide cleavage sites did not release GFP from HC-Pro, but efficiently expressed GFP as dense fluorescent aggregates. However, inserting 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.