Progress 06/25/07 to 05/21/12
Outputs
Progress Report 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. 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. We completed the microarray analysis of gene expression in strain B728a, and derivatives containing mutations in three of the Gac pathway genes that encode regulators required for disease on snap beans 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. We discovered that the Gac transcriptome in B725a includes over 1200 genes. 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 levels. The choice of carbon source did not alter gene expression significantly. We established that the GacS and GacA genes truly function as a regulatory pair and that GacA does not regulate gene expression independent of GacS. That we can detect artifacts is important because we know that among the many genes regulated by Gac directly are genes important for brown spot disease. Our microarray analysis of a 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 have continued our microarray of the effects of growth conditions on gene expression. We have completed preparation of ribonucleic acids (RNA) samples from B728a grown on solid media and in liquid media (planktonic growth) and that RNA is currently being analyzed by microarray by the vendor. We developed 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, transgenic tomato plants expressing a protein that inhibits spread of the virus by the thrips insect vector have been constructed. Our collaborators were not able to construct transgenic potato but were able to construct transgenic tomato plants that express the GNs protein. These plants are currently being accessed for inhibition of TSWV transmission by thrips. These improved methods will aid in our analysis of viral replication in the plant and the insect vector and provide novel strategies for inhibiting the spread of these two viruses. Accomplishments 01 Iron is required for food contamination by Salmonella enterica. ARS scientists in collaboration with researchers in the Department of Plant Pathology at the University of Wisconsin-Madison discovered a Salmonella gene called aroA that is needed by the bacterium to grow on alfalfa seedlings. This gene controls bacterial growth by enabling the producti of a bacterial substance that scavenges iron from the alfalfa seedlings and is required for the growth of Salmonella on lettuce, another fresh produce associated with salmonellosis outbreak. If we can disrupt the uptake of iron by Salmonella during plant growth, we can reduce or eliminate contamination of our food by this organism. We are currently extending this study to analyze the impact of aroA in plant colonization by the bean brown spot pathogen Pseudomonas syringae.
Impacts
(N/A)
Publications
- Frost, K., Willis, D.K., Groves, R. 2011. Detection and variability of Aster Yellows Phytoplasma Titer in its insect vector, Macrosteles quadrilineatus (Hemiptera: Cicadellidae). Journal of Economic Entomology. 104(6):1800-1816.
- Marquez, M., Weber, B., Witherell, A., Willis, D.K., Charkowski, A.O. 2011. The 3-hydroxy-2-butanone pathway is required for Pectobacterium carotovorum pathogenesis. PLoS One. Available:
- Hao, L., Willis, D.K., Andrews-Polymenis, H., Mcclelland, M., Barak, J.D. 2012. Requirement of siderophore biosynthesis for plant colonization by Salmonella enterica. Applied and Environmental Microbiology. 78(13):4561- 4570.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. Accomplishments 01 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 molecule insect attractants, and, in other bacterial species, affect virulence an fitness. Disruption of the budB gene reduced virulence of P. c. subsp. carotovorum on potato tubers and impaired the ability of the bacterium t 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, turn, maximizes the activity of the Pectobacterium pectate lyases. It i the pectate lyase enzymes that rot potato tissue. This identifies contr of pH as a target for reduction of this disease during potato storage. high pH can be prevented during storage, then loss of potatoes due to so rot will be reduced. 02 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-amyla 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 Bmy protein from having a significant contribution to the overall beta-amyla 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.
Impacts
(N/A)
Publications
- Vinje, M.A., Willis, D.K., Henson, C.A., Duke, S.H. 2011. Differential expression of two �-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.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report 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. 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 GacS-GacA genetic regulon controls the expression of a variety of important virulence functions in the snap bean pathogen Pseudomonas syringae pv. syringae B728a, to the degree that mutation of either the gacS or the gacA gene results in a complete loss of pathogenicity. Microarray analysis demonstrated that 21% of the total genes (1100 of 5137 predicted genes) of P. s. syringae strain B728a was controlled by gacA/gacS. A bacterial strain containing a mutation in the gacS/gacA- regulated salA gene shows a similar expression profile to the B728a parent, except in gene regions involved in the production of three bacterial toxins. Since we previously established that salA mutants are unable to cause disease symptoms on bean, this implicates these three toxins as significant contributors to the disease process. Recent experiments indicated that bacterial swarming, a phenotype that has been associated with virulence and is also regulated by the Gac regulon, does not require bacterial growth. We identified fundamental differences in gene expression between liquid grown bacteria and bacteria grown in colonies on solid media. Both swarming and micro-colony formation are expected to be contributors to bacterial fitness on plants. We successfully quantified the Tomato spotted wilt virus (TSWV) transcripts encoding the nucleocapsid protein (N) and the non-structural protein (NSs) within infected host plants using real-time reverse transcription polymerase chain reaction (RT-qPCR.) High efficiency primers specific for these messenger ribonucleic acids (RNAs) were developed and used for the experiments. Our results indicate the N gene is expressed approximately 5-fold higher than the NSs gene in infected plants. This result set up the analysis of the expression of these two genes and the remaining three TSWV genes within Western Flower Thrips, the agronomically important vector of TSWV. Our efforts will confirm that TSWV infects and actively replicates within the insect vector. This information is vital for formulating novel control strategies for the acquisition & spread of this virus. We improved methods to analyze RNA produced by Maize fine streak virus (MFSV) within infected maize tissue using real-time RT-qPCR. We established that MFSV virion RNA is approximately 50-fold more abundant than the replicative virion-complementary RNA in symptomatic maize leaf tissue. The abundance of the MFSV gene transcripts decreased with distance from the 3� promoter suggesting transcription in infected maize similar to transcription in the well studied rhabdovirus Vesticular stomatitis virus (VSV). Our research discovered that two MFSV specific transcripts, not present in VSV or other animal Rhabdoviruses, accumulated to higher levels than predicted suggesting that these transcripts are produced by an alternate transcription process or exhibit increased stability relative the remaining MFSV transcripts. This result not only illustrates differences in the expression of plant-infecting Rhabdoviruses compared to their animal-infecting relatives, but also provides potential targets for inhibition of the replication of MFSV in its agronomically important host, corn. Accomplishments 01 Identification of molecular targets for juvenile hormone action in insec 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 analys to identify specific genes influenced by juvenile hormone. Our microarra analysis revealed relatively few insect genes were verifiably altered in their expression by addition of juvenile hormone. Two genes that showed increased expression were of unknown function. Expression of the Epac ge involved in insect development was increased four-fold in the presence o juvenile hormone. Identification of Epac and additional juvenile hormone influenced genes provide novel targets for genetic and chemical control insect pests. 02 Quantification of the aster yellows phytoplasma within its insect vector The aster yellows phytoplasma is transmitted by the aster leafhopper, or Macrosteles quadrilineatus. Aster yellows is an agronomically important pathogen of carrot. ARS scientists in the Vegetable Crops Research Unit collaboration with university researchers in the Department of Entomolog at the University of Wisconsin-Madison developed a quantitative real-tim reverse transcription polymerase chain reaction PCR (qPCR) assay to measure aster yellows concentration in insect deoxyribonucleic acid (DNA extracts. Our results demonstrated that the aster yellows phytoplasma efficiently replicates within the leafhopper reaching a maximum titer in 10 days. This new technique will enable us to examine the biological factors governing aster yellows replication in the leafhopper and examin if aster yellows population size is associated with the frequency of transmission. 03 Determination of the regulation of �-amylase within high and low activit malting barley. �-amylase is an enzyme important to the malting and brewing industry. The objective of this study was to determine the genet basis of differences �-amylase activity in barley (Hordeum vulgare L.). ARS scientists in the Vegetable Crops Research Unit and the Cereal Crops Research Unit in collaboration with university researchers in the Department of Agronomy at the University of Wisconsin used real-time RT- qPCR and protein activity analysis to investigate basis for differing �- amylase activities among four barley cultivars. Our results demonstrated that cultivars with high �-amylase activity also had higher messenger ribonucleic acid (mRNA) levels suggesting increased gene expression is t source of the increased �-amylase activity. This increase was not linked to known genetic markers within the �-amylase gene. We also found that total protein was higher in grains from high �-amylase cultivars. Higher levels of total protein could also contribute to the higher levels of �- amylase activity.
Impacts
(N/A)
Publications
- Willis, D.K., Wang, J., Stanford, J.R., Orth, A., Goodman, W.G. 2010. Microarray Analysis of Juvenile Hormone Response in Drosophila melanogaster S2 cells. Journal of Insect Science. 10(6). Available: http://www.insectscience.org/10.66/i1536-2442-10-66.pdf.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations 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.
Impacts
(N/A)
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, and a High Voltage HEPES Buffer System. 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.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations 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).
Impacts
(N/A)
Publications
- 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.
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