ARS | Publication request: Elucidating the Potential of Plant Rhabdoviruses as Vector Expressions Systems

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 25, 2009
Publication Date: August 1, 2009
Citation: Cisneros, F.M., Redinbaugh, M.G., Tsai, C.W., Whitfield, A.E., Hogenhout, S.A. 2009. Elucidating the Potential of Plant Rhabdoviruses as Vector Expressions Systems [abstract]. North Central American Phytopathological Society Meeting, June 25-26, 2009, Iowa State University, IA.

Technical Abstract: Maize fine streak virus (MFSV) is a member of the genus Nucleorhabdovirus that is transmitted by the leafhopper Graminella nigrifons. The virus replicates in both its maize host and its insect vector. To determine whether Drosophila S2 cells support the production of full-length MFSV proteins, we inserted the nucleoprotein (N), phosphoprotein (P) and replicase protein (L) ORFs of MFSV upstream of the V5 epitope/ 6X His tag of the pMT/V5-His-Topo vector. The S2 cells were transfected with these plasmid DNAs. When analyzed by western blot, antibodies to the V5 epitope clearly reacted with proteins of ~55 and 43 kDa in cells transfected with plasmids carrying the N and P genes, respectively, the sizes expected for the full-length fusion proteins. No bands were detected in non-transfected Drosophila S2 cells. The expression of the N gene was also tested with antibodies raised against MFSV virions, which detect the N protein as well as several other virus proteins. MFSV virion antibodies detected a protein of ~55 kDa in S2 cell protein extracts. Antibodies raised against a peptide sequence from the deduced MFSV P protein reacted with a protein of ~ 43 kDa in transfected S2 cell protein extracts. The MFSV N and P genes were detected over a period of 4 days after induction of gene expression with Cu++, but were not detected in cells not exposed to Cu++. Experiments are underway to asses MFSV L gene expression. Our results suggest that the the full-length MFSV N and P proteins can be stably expressed in S2 cells.