Location: Vegetable Crops Research2008 Annual Report
1a. Objectives (from AD-416)
The long-term objective of this project is to develop an improved understanding of the genetics of bacterial and viral pathogens that cause disease on snap bean, tomato and potato. Over the next 5 years we will focus on the following objectives: Objective 1: Use P. syringae pv. syringae B728a genomic expression chips to identify and characterize genes regulated by the gacS/gacA two-component regulatory system. Sub-objective 1.A. Use genomic expression chips to identify the members of the gacA/gacS transcriptome that are regulated under a variety of growth conditions. Sub-objective 1.B. Functional genomic analysis of gacS/gacA regulated genes. Objective 2: Develop and analyze transgenic plants expressing a viral protein that may inhibit Tomato spotted wilt virus (TSWV) transmission by thrips. Sub-objective 2.A. Develop real-time RT-PCR methodologies to quantitate TSWV replication in host plants and the thrips vector. Sub-objective 2.b. Construct and characterize transgenic tomato plants expressing the TSWV glycoprotein GN-S.
1b. Approach (from AD-416)
For Objective 1: Bacterial growth conditions that will be analyzed include varying pH, iron availability and liquid vs. solid media. These growth conditions are all known to affect the growth of bacteria on plants. High quality RNA will be prepared using standard bacterial protocols. RNAs will be used to probe commercially available genomic expression arrays containing oligo DNA markers for all 3,840 genes within the B728a genome. Reproducibility will be ensured by having standardized hybridization protocols performed by the vendor, with the chip data processed by the SY using proprietary software. Changes in gene expression will be confirmed using real-time RT-PCR. Genes that show differential expression under the various growth conditions will be mutated and their effect on plant virulence determined. For Objective 2: All three TSWV RNA contain very similar but not identical sequences at their ends. We will use these end sequences to design primers that are specific to either the genomic RNA (contained in the viral particle) or anti-genomicRNA (necessary for replication) to produce cDNA specific to that RNA. We will determine the amounts of viral message RNA species by using random hexamers to generate cDNA. The viral RNA within each cDNA will be quantitated by real-time PCR using our standard protocols. The amount of each RNA species will be determined by using a standard curve consisting of a dilution series of cloned viral DNA of known concentration. As a preliminary to the analsysis of TSWV, we will determine the relative amounts of genomic, anti-genomic, and viral mRNAs expressed by the maize pathogen Maize fine streak virus. MFSV is a mono-partite negative-sense virus that contains only a single RNA genome and avoids the complexity of distinquishing three RNA genomes containing related sequences as is the case with TSWV. We have shown that feeding thrips a modified form of the TSWV glycoprotein GN (designated GN-S) dramatically inhibits the acquisition of the virus and the ability of the thrips to transmit the virus. This most likely is due to the saturation of viral binding sites within the thrips guts by GN-S thus preventing viral binding and transport of the TSWV virion through the intestinal lining. We will express the GN-S protein in potato and other hosts to establish that this protein can inhibit the acquisition and transmission of TSWV when expressed within the plant. The GN-S ORF will be cloned into an Agrobacterium vector. This construct will be either transiently expressed using an Agro launching technique or transformed into a susceptible host. Plants will be analyzed for GN-S gene expression using real-time RT-PCR and GN-S protein expression by western blot. Thrips will be fed on transiently expressing leaf discs or transformed plants showing a high level of expression of the GN-S protein for a two hour acquisition period and then moved to TSWV infected hosts. Acquisition of TSWV by thrips will be analyzed using real-time RT-PCR and transmission of TSWV to host plants will be quantitated using a leaf disc or green house assay.
3. Progress Report
Arthropod vectors play an essential role in dissemination of viruses that cause diseases in humans, animals, and plants. Tomato spotted wilt virus (TSWV) is transmitted in a persistent propagative manner by its thrips vectors. In concomitant and sequential feeding experiments, a soluble form of a TSWV membrane protein significantly reduced transmission by thrips. These results are the first to show that insect transmission of a membrane-bound virus can be blocked through inhibition of viral acquisition. Application of this approach to arthropod transmitted viruses of animals and humans has profound implications for the development of therapeutic and dissemination intervention strategies. RNA integrity is critical for successful real-time qRT-PCR RNA quantitation. The level of integrity required for successful quantitation has not been determined for bacterial RNA. Three RNA isolation methods were evaluated for their ability to produce high-quality RNA from D. dadanti. A hot SDS-hot phenol RNA method gave the highest RNA quality and required only two DNase treatments to remove DNA. The assessment of RNA integrity was critical for obtaining meaningful gene expression data. RIN values below 7.0 resulted in high variation and loss of statistical significance when gene expression was analyzed by real-time qRT-PCR. We found that RNA preparations of different quality yielded drastic differences in relative gene expression ratios and led to major errors in the quantification of transcript levels. This work provides guidelines for RNA isolation and quality assessment that will valuable for gene expression studies in a wide range of bacteria. Efforts to analyze the replicative RNA produced by Maize fine streak virus (MFSV) within maize tissue was complicated by the lack of specificity during cDNA generation using standard reverse transcriptase protocols. Real-time qRT-PCR using cDNA generated by priming with random hexamers does not distinguish between virion (vRNA) and virion-complementary (vcRNA) or viral mRNA. Detection and quantitation of the products of viral replication requires strand-specific cDNA synthesis. However, auto-priming (generation of cDNA without primers) of the vRNA or vcRNA and false priming of the incorrect strand complicate detection and quantitation of viral replicative RNAs. We have identified efficient primers specific to each of the seven MFSV ORFs as well as vRNA and vcRNA. Strand-specificity was improved by increasing cDNA reaction temperature from 42°C to 60°C with tagged primers which reduced auto-priming 23-fold and non-specific priming by 21 to 315-fold. Using this methodology, we established that MFSV vRNA is 30 to 60-fold more abundant than the replicative vcRNA in maize leaf tissue exhibiting fine streak symptoms. In contrast to mRNA ratios established in the well studied rhabdovirus Vesticular stomatitis virus, the N, P and L gene messages of MFSV were statistically equivalent in symptomatic maize leaf tissue. This research is relevant to National Progam/Component (NP 303/component 2).
1. Inhibition of Tomato spotted wilt virus transmission by thrips. Tomato spotted wilt virus (TSWV) is transmitted in a persistent propagative manner by its thrips vectors. In concomitant and sequential feeding experiments, a soluble form of a TSWV membrane protein significantly reduced virus transmission by thrips. These results are the first to show that insect transmission of a membrane-bound virus can be blocked through inhibition of viral acquisition. Application of this approach to arthropod transmitted viruses of animals and humans has profound implications for the development of therapeutic and dissemination intervention strategies. This research is relevant to Component 2 of the NP 303 Action Plan.
5. Significant Activities that Support Special Target Populations
Whitfield, A.E., Kumar, N., Rotenberg, D., Ullman, D.E., Wyman, E.A., Zietlow, C., Willis, D.K., German, T.L. 2008. A soluble form of the tomato spotted wilt virus (TSWV) glycoprotein GN (GN-S) inhibits transmission of TSWV by Frankliniella occidentalis. Phytopathology. 98:45-50.