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United States Department of Agriculture

Agricultural Research Service


Location: Vegetable Crops Research Unit

2009 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
The GacS/GacA genetic regulatory network, first described in our laboratory, controls significant aspects of bacterial disease biology in both animal and plant hosts. In the snap bean pathogen Pseudomonas syringae pv. syringae, these genes modulate fundamental virulence factors, such as the production of toxins and bacterial fitness in the field environment, to the degree that mutation of either the gacS or gacA gene leads to the complete loss of pathogenicity. We have developed methods for the reliable isolation of high quality Ribonucleic acid (RNA)from both liquid- and plate-grown bacteria, enabling us to use microarray technology to study the effects of gacS/gacA regulation under varying growth conditions. Initial results indicate that the gacS/gacA regulatory network or "regulon" contains as much as 21% of the total genes (1100 of 5137 predicted genes) of P. s. syringae strain B728a under a given set of conditions. Comparisons of gacS or gacA mutants to each other showed very high consistency in their expression profiles indicating that both genes participate equally in the regulon. Changing growth conditions altered the expression profiles of both wild-type and mutant bacteria, while still demonstrating that the gacS/gacA genes control a large number of chromosomal genes under all conditions examined. This work provides a scaffold for the analysis of this important bacterial regulon required for plant disease and fitness.

Efforts were initiated to analyze the replicative RNA produced by Tomato spotted wilt virus (TSWV) within the plant host and the insect vector (western flower thrips) with the construction of a clone containing the entire TSWV small RNA (sRNA). This viral RNA encodes the nucleocapsid protein (N) and the small non-structural protein (NSs). This construct contains ribozymes flanking the sRNA sequence so that in vitro generated RNA will be cleave into a precise viral sRNA genome. This clone is essential for the analysis of cDNA primers constructed to specifically detect TSWV sRNA transcripts. High efficiency primers for real-time RT-qPCR quantification of N and NSs mRNA have been designed and successfully tested.

Arthropod vectors play an essential role in dissemination of viruses that cause diseases in humans, animals, and plants. TSWV is transmitted in a persistent propagative manner by Frankliniella occidentalis, the western flower thrips. Real-time qPCR using primers specific for the TSWV N gene were used to establish TSWV titer within individual insects. The data support the hypothesis that a viruliferous thrips is more likely to transmit multiple times if it harbors a high titer of virus. Male thrips were more efficient at transmitting TSWV multiple times compared with female thrips of the same cohort. However, females harbored two to three times more copies of TSWV-N RNA per insect, indicating that factors other than absolute virus titer in the insect could contribute to a successful transmission event. This quantitative relationship provides new insights into the biological parameters that may influence the spread of TSWV by thrips.

1. Novel resistance to bacterial speck detected in field grown tomato. ARS scientists in the Vegetable Crops Research Unit in Madison Wisconsin described inhibition of bacterial speck populations by the resistant Rio Grande-Pto tomato cultivar under field conditions. Pto resistance to Pseudomonas syringe pv. tomato was previously thought to involve the recognition of pathogen effector proteins within the plant cell. This research demonstrates that Pto resistance also functions at the leaf surface reducing spray-inoculated bacterial populations to undetectable levels on Pto-resistant plants. The result may explain the relative long period of effectiveness of Pto resistance in the field. Loss of the avrPto avirulence determinant from the pathogen did not alleviate field resistance although increased growth and lesion formation on resistant plants was noted under growth chamber conditions. This research significantly contributes to our understanding of the genetic mechanisms that lead to durable plant resistance under field conditions.

2. Identification of molecular targets for juvenile hormone action in insects. The insect juvenile hormones represent a family of molecules that regulate a diversity of processes in the insect life cycle. Juvenile hormone affects insect development by maintaining the larval stage and inhibiting metamorphosis, and it is this feature that has led to the development of juvenile hormone analogs and agonists as agronomically important insecticides. ARS scientists in the Vegetable Crops Research Unit in collaboration with university researchers in the Department of Entomology at the University of Wisconsin-Madison used microarray analysis to identify specific genes influenced by juvenile hormone. Chief among these genes was Epac a known insect developmental regulator. Identification of Epac and additional juvenile hormone influenced genes provide novel targets for genetic and chemical control of insect pests.

3. Analysis 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 caused extensive damage to lettuce in Hawaii with crop losses of 50 – 90%, crippling the vegetable industry. The ARS Vegetable Crops Research Unit in collaboration with researchers in the Department of Plant Pathology at Kansas State University and the University of Wisconsin-Madison, explored the relationship between virus titer in adult thrips and transmission. It was discovered that although male thrips contain less virus than female thrips, they transmit the virus at a higher frequency and that generally a high amount of virus within an individual insect was necessary for efficient repeated transmission of the pathogen. The findings suggest that virus titer in adult thrips may be a reliable indicator of TSWV incidence under field conditions.

4. A bacterial movement gene is required for virulence on plants. Dickeya dadantii (formerly Erwinia chrysanthemi) causes wilting and soft rot in a wide range of plants, including ornamental plants and economically important vegetable crops. D. dadantii produces a variety of cell wall-degrading enzymes, such as pectate lyases, polygalacturonases, and cellulases. These enzymes cause plant tissue maceration and are the primary pathogenicity factor of this pathogen. The ARS Vegetable Crops Research Unit in collaboration with scientists in the Department of Plant Pathology at the University of Wisconsin-Madison discovered that a gene that regulates bacterial movement also controls the expression of pectate lyase by this bacterium. In addition, loss of this genetic locus reduces biofilm formation and virulence on the plant host. Unraveling the multiple mechanisms and signals used to co-regulate motility and virulence proteins will enable new control methods for plant pathogenic bacteria.

5. Determination of the effect of RNA quality on the validity of real-time quantitative PCR. Real-time RT-qPCR is now recognized as the most accurate method of quantifying RNA transcripts with bacterial cells. ARS scientists in the Vegetable Crops Research Unit collaborated with scientists in the Department of Plant Pathology at the University of Wisconsin-Madison to demonstrate the impact of Ribonucleic acid (RNA) quality on the accuracy of this important analytic methodology. Several RNA isolation methods were evaluated using micro-fluidic capillary electrophoresis to determine their effect on RNA quality. Subsequently, RNA of various qualities were assessed for their effect on the accuracy and reproducibility of real-time RT-qPCR quantification. Only the highest quality RNA was found to be acceptable for the accurate determination of RNA transcript amount. RNA preparations of lower 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 be valuable for gene expression studies in a wide range of bacteria.

Review Publications
Hirano, S.S., Kinscherf, T.G., Upper, C.D., Willis, D.K. 2009. Population Dynamics of Pseudomonas Syringae pv. Tomato Strains on Tomato Cultivars Rio Grande and Rio Grande-Pto Under Field Conditions. Journal of Phytopathology. 157(4):219-227.

Wang, J., Lindholm, J.R., Willis, D.K., Orth, A., Goodman, W.G. 2009. Juvenile Hormone Regulation of Drosophila Epac - A Guanine Nucleotide Exchange Factor for Rap1 Small GTPase. Molecular and Cellular Endocrinology. 305(1-2):30-37.

Rotenberg, D., Krishna Kumar, N.K., Ullman, D.E., Montero-Astua, M., Willis, D.K., German, T.L., Whitfield, A.E. 2009. Variation in Tomato Spotted Wilt Virus Titer in Frankliniella Occidentalis and Its Association with Frequency of Transmission. Phytopathology. 99(4):404-410.

Kinscherf, T.G., Yap, M.N., Charkowsky, A.O., Willis, D.K. 2009. CHEF Procedures: A Rapid High-temperature Method for Sample Preparation, A High Voltage Hepes Buffer System and the Use of Nusieve® Agarose. Journal of Rapid Methods and Automation in Microbiology. 17(1):9-16.

Jahn, C.E., Charkowski, A.O., Willis, D.K. 2008. Evaluation of isolation methods for bacterial RNA quantitation in Dickeya dadantii. Applied and Environmental Microbiology. 75(2):318-324.

Last Modified: 4/18/2014
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