2012 Annual Report
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.
This is the final report for project 3655-22000-019-04S that terminated in June 2012. Under this Specific Cooperative Agreement (SCA), researchers developed methods to analyze viral replication in plants and insect vectors. Tomato spotted wilt virus (TSWV) is considered one of the ten most devastating plant viruses. TSWV is transmitted by western flower thrips, an insect vector that is widespread and 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. We also studied Maize fine streak virus (MFSV) an important pathogen of corn in the United States. We developed methods to analyze the replication of TSWV and MFSV within infected plants and insects. We developed real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) primers that are specific for the messenger ribonucleic acids (RNA) of MFSV and we are able to measure the replicative RNA of the virus independent of transcription. We demonstrated that MFSV expresses its genes very differently in plants than the related viruses from animals. Two MFSV genes not found in related animal viruses were highly expressed in infected corn and these two genes are now prime targets for creating virus resistant plants. Our investigations into TSWV were very productive. We developed methods that allow the real-time RT-qPCR analysis of gene expression within a single thrips. We showed that male thrips actually transmit TSWV at a higher frequency than female thrips even though female thrips contain a higher number of viruses. We established that feeding thrips the viral protein GN inhibits the spread of the virus by the insect. Feeding thrips the GN protein apparently masks the viral binding sites in the insect’s intestines. This is an important discovery and provides a unique approach to control the spread of viral diseases of plants. In initial experiments, the GN-expressing transgenic tomato plants appear to inhibit the insect acquisition and spread of TSWV.
The methodologies developed under this SCA had impacts outside of the specific area of study. Collaborations with researchers led to numerous investigations and publications in areas such as: establishing the mode of action of hormone-based insecticides; improving malting barley, determining the genetic source of the orange color of carrots; studying gene expression in bacterial pathogens of snap beans and potatoes; and discovering genes that are needed by bacteria that cause food poisoning to contaminate our fresh vegetables.
This research relates to Objective 2, Develop and analyze transgenic plants expressing a viral protein that may inhibit TSWV transmission by thrips.