1a.Objectives (from AD-416)
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.
1b.Approach (from AD-416)
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.
Inhibition of virus transmission by western flower thrips - tomato spotted wilt virus (TSWV) is considered one of the ten most devastating plant viruses. TSWV and its insect vectors thrips are widespread and as a result, they cause extensive damage to agronomic crops. For example, TSWV has caused extensive damage to lettuce in Hawaii with crop losses of 50 – 90%, crippling the vegetable industry. In collaboration with researchers, methodologies to quantify viral messenger ribonucleic acid (mRNA) in plants and insects were developed. We improved methods to analyze RNA produced by Maize fine streak virus (MFSV) and Tomato spotted wilt virus (TSWV) within infected plants. We confirmed the over-expression of two plant virus-specific transcripts from MFSV infected corn and showed that expression was elevated early in the infection process. These two MFSV transcripts are now prime targets for transgenic virus resistance. In collaboration with researchers, transgenic tomato plants expressing the TSWV transmission inhibiting protein GNs have been constructed. Work is underway to maximize protein expression and evaluate the efficacy of this approach in preventing the insect acquisition and spread of TSWV. The project is monitored through in person discussions and e-mail exchanges.