2011 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.
The bacterium Pseudomonas syringae pv. syringae causes brown spot disease of snap bean and is an agronomically important pathogen in Wisconsin and other snap-bean producing areas of the United States. Using Pseudomonas syringae pv. syringae strain B728a, we completed the microarray analysis of genome-wide gene expression of, and mutant derivatives containing mutations in three of the Gac pathway genes (gacS, gacA, and salA). The Gac pathway genes encode regulators that are required for disease on snap bean in the field. The analysis included growth under four conditions: dextrose or glycerol as a carbon source, and two levels of casamino acids (nitrogen) in the medium. The Gac transcriptome in B725a includes over 1200 genes regulated by Gac. We discovered a significant artifact caused by growth conditions, as a number of genes that appeared to be negatively regulated by Gac were actually induced by high casamino acid (nitrogen) levels. In contrast, the choice of carbon source did not alter gene expression significantly. It should be noted that the expression profiles of mutations in gacS or gacA were virtually identical indicating that GacS and GacA truly function as a regulatory pair and that GacA does not regulate gene expression independent of GacS. Our discovery of a significant artifact caused by growth conditions is important because we know that among the many genes regulated by Gac directly, there are genes important for brown spot disease. For example, our microarray analysis of the salA mutant combined with mutational analysis of the bacterium identified the toxins syringopeptin and syringolin A as major contributors to brown spot disease symptom development on bean. This result establishes that breeding toxin resistance plants could provide a novel control method for brown spot disease of snap bean.
We improved methods to analyze viral replication of Maize fine streak virus (MFSV) and Tomato spotted wilt virus (TSWV) within infected plants. MFSV is an important pathogen of corn in the United States while TSWV infects many agronomically important crops including tomato, lettuce, and pineapple. Unlike current methods of detection, our methods can distinguish between active viral infections and the mere presence of the virus in infected tissues. We confirmed the over-expression of two plant virus-specific transcripts from MFSV within 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 at UW-Madison (SCA) and Kansas State University, transgenic tomato plants expressing a protein that inhibits spread of the virus by the thrips insect vector 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. Together, these improved methods will aid in our analysis of viral replication in plant and the insect vector and provide novel strategies for inhibiting the spread of these two viruses.
The level of beta-amylase influences the usefulness of the grain. In barley, the level of beta-amylase influences the usefulness of the grain for malting and affects its market value. A higher amount of beta-amylase in the grain increases the value of the crop. In collaboration with ARS researchers in Madison, Wisconsin and University of Wisconsin-Madison scientists, we analyzed the two barley beta-amylase genes (Bmy1 and Bmy2) in four types of barley. The Bmy1 protein was far more prevalent than Bmy2 at all developmental stages in all types of barley. Low levels of Bmy2 observed in the developing and mature grain likely preclude the Bmy2 protein from having a significant contribution to the overall beta-amylase activity in the developing and mature grain. By far most of the beta-amylase present in both the developing and mature grain is Bmy1. This information is important to barley breeders in their efforts to increase beta-amylase activity. Their focus should be concentrated in increasing the amount of Bmy1 in mature barley grain.
Microarray analysis of a bacterial potato pathogen. Pectobacterium species are bacterial pathogens that cause soft rot diseases in potatoes and several other crops worldwide. Pectobacterium carotovorum subsp. carotovorum is a major contributor to loss of potatoes in storage. From the combined efforts of ARS scientists in Madison, Wisconsin and researchers in the Department of Plant Pathology at the University of Wisconsin-Madison, we verified the microarray analysis of the potato pathogen and identified a gene, budB, that was expressed at a significantly higher level in potato tubers compared to potato stems. This gene controls the expression of volatile compounds produced by the bacterium that have been shown to act as plant growth promoting molecules, insect attractants, and, in other bacterial species, affect virulence and fitness. Disruption of the budB gene reduced virulence of P. c. subsp. carotovorum on potato tubers and impaired the ability of the bacterium to alter potato tubers in ways that would enhance growth of the pathogen. The budB gene is required for raising the pH of the potato tissue that, in turn, maximizes the activity of the Pectobacterium pectate lyases. It is the pectate lyase enzymes that rot potato tissue. This identifies control of pH as a target for reduction of this disease during potato storage. If high pH can be prevented during storage, then loss of potatoes due to soft rot will be reduced.
Vinje, M.A., Willis, D.K., Henson, C.A., Duke, S.H. 2011. Differential expression of two ß-amylase genes (Bmy1 and Bmy2) in developing and mature barley grain. Planta. 233(5):1001-1010.
Vinje, M.A., Willis, D.K., Duke, S.H., Henson, C.A. 2010. Differential RNA expression of Bmy1 during seed development and the association with beta-amylase accumulation, activity, and total protein. Plant Physiology and Biochemistry. 49:39-45.