Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: May 3, 2010
Publication Date: June 1, 2010
Citation: Tatineni, S., French, R.C. 2010. GFP is Efficiently Expressed by Wheat Streak Mosaic Virus Using a Range of Tritimovirus NIa Cleavage Sites and Forms Dense Aggregates in Cereal Hosts. American Phytopathological Society Annual Meeting. Phytopathology Volume 100, Page S125. Technical Abstract: Wheat streak mosaic virus (WSMV)-based transient expression vector was developed to express GFP as a marker protein. The GFP cistron was engineered between the P1 and HC-Pro cistrons in an infectious cDNA clone of WSMV. The cleavage sites, P3/6KI, 6KI/CI, NIa/NIb, or NIb/CP, from WSMV were fused to the C-terminus of GFP such that free GFP will be released after proteolytic processing of viral polyprotein. WSMV-GFP constructs infected wheat similar to that of the wild-type virus and expressed GFP mostly as aggregated structures even though proteolytically processed free GFP was detected by immuno-blots. GFP was similarly expressed as aggregates with heterologous cleavage sites from Brome streak mosaic virus or by mutating the -1 amino acid (aa) position of WSMV cleavage sites to either alanine or arginine. Binary vectors with GFP cistron containing aa that would result from cleavage sites (GFP-CS) or co-infiltration of GFP-CS with WSMV NIa cistron failed to form such aggregates in Agrobacterium-infiltrated Nicotiana benthamiana leaves, suggesting that neither aa that result from cleavage nor possible interactions between the NIa-pro and GFP-CS are involved in the formation of aggregated GFP. However, GFP was expressed mostly as free protein from WSMV-GFP with Foot-and-mouth disease virus 2A peptide (33 aa) at the C-terminus of GFP cistron. WSMV-GFP vectors were relatively stable in wheat plants and expressed GFP beyond five serial passages at 14-day intervals.