DEVELOP AND ANALYZE PLANTS EXPRESSING A VIRAL PROTEIN THAT MAY INHIBIT TSWV TRANSMISSION BY THRIPS
Project Number: 3655-22000-019-04
Specific Cooperative Agreement
Start Date: Aug 27, 2007
End Date: Jun 30, 2012
The first objective of this cooperative research project is to quantitate the viral RNAs produced by the monopartite negative-sense virus Maize fine streak virus (MFSV). This analysis will yield the first description of the transcription strategy of this recently describe Rhabdovirus within the plant host. The second objective of this cooperative research is to construct plasmids containing the Tomato spotted wilt virus (TSWV) GN-S that can be used for transient expression in plants. We have shown that the GN-S protein inhibits the acquisition and transmission of TSWV by thrips when administered in an artificial feeding assay. This research will establish if insect transmission inhibition will occur when the GN-S protein is expressed within a plant host. If successful, these experiments will serve as a proof-of-concept for a novel approach to limit the extent of viral transmission by an insect vector.
RNA will be extracted from MFSV viral particles isolated from infected plants for use as a standard curve for normalization real-time RT-PCR. Total mRNA from infected plants will be used to quantitate MSFV genomic and mRNA in plants using real-time RT-PCR. Specific primers will be used in the cDNA reaction to distinguish MFSV mRNA, genomic, and anti-genomic species. The GN-S ORF will be cloned behind a 35S promoter in the Agrobacterium shuttle vector pCAMBIA. The clones will be transformed into Agrobacterium and used in transient expression assays. Plant leaves will be infiltrated with Agrobacteria and the expression of GN-S will be monitored using real-time RT-PCR and western blots to establish a time course of expression. Leaf discs from infiltrated areas will be used to feed thrips prior to their exposure to TSWV infected tissue. Acquisition of TSWV by thrips will be monitored using real-time RT-PCR. Transmission of TSWV will be ascertained using a leaf-disc assay using ELISA for viral detection.