Progress 06/29/07 to 04/23/12
Outputs
Progress Report Objectives (from AD-416): 1. Identify plant genes whose pattern of expression changes in resistant compared to susceptible cereals when inoculated with viral or fungal pathogens. The research emphasis will be on barley and cereal yellow dwarf viruses, Fusarium head blight (scab), and leaf rust. 2. Develop a virus-induced gene silencing system (VIGS) as a tool to determine gene function in cereal crops. 3. Use the VIGs system to determine which cereal genes identified are essential in resistance and susceptibility responses (resistance pathway components). 4. Develop useful DNA markers for improvement of viral and fungal resistance. a. Develop markers for the genes verified to encode resistance pathway components in for use in Marker-Assisted Selection (MAS). b. Generate and characterize SSR-enriched libraries from oat. c. Identify DNA markers linked to the wheatgrass-derived Bdv3 and FHB resistance for MAS in wheat. Approach (from AD-416): Barley and cereal yellow dwarf viruses (YDV) are causing significant losses to small grain production. Fungal pathogens, such as Fusarium head blight and cereal rusts continue to cause major damage and losses. Development of genetic resistance in small grains to these pathogens is hindered by the lack of information about mechanisms that confer host- plant resistance and the lack of linked DNA markers to assist in the development of improved germplasm. Our approach will be to use new and existing molecular genetic methods such as gene expression arrays, transient gene silencing and to develop an improved understanding of the genetic and biochemical mechanisms that can confer host-plant resistance disease resistance and utilize this information to develop new strategies for enhancing genetic resistance to cereal viral and fungal diseases. This is the final report for this project. For further information, please see 3602-21220-013-00D. The primary objectives of this work were the development of cereals with improved resistance to Barley yellow dwarf virus (BYDV) and Yellow dwarf virus (YDV), the development of markers that could be used to map BYDV and YDV resistance in oats, and development of diagnostic tools for common cereal viruses. Over the course of this project significant progress was made in all of these areas. Perhaps most significant in these efforts was the introgression of resistance to YDV, crown rust and stem rust from wild oat into cultivated oats. Four cycles of backcrossing to elite oat lines have been performed and advanced material is now available with strong resistance to all three pathogens. Additionally, a PCR-based assay was developed that can detect up to ten common wheat viruses, including BYDV and YDV. This assay is now being adopted by many of the plant diagnostic laboratories in the cereal production areas of the US and Canada. The central objective for this project was to develop methods that would permit identification of genes with essential functions in wheat�s resistance to Fusarium head blight (FHB) and other significant diseases of wheat. A method for virus-induced gene silencing (VIGS) was developed that reliably knocks-down expression of genes expressed in wheat. Prior to the development of this technique it was extremely difficult to assess the function of wheat genes. This difficulty is largely due to the fact that all wheat that is grown for food production is polyploid, meaning that it has multiple sets of chromosomes. This prevents conventional genetic analysis based on inactivation of genes by mutations, because wheat almost always has multiple copies of each gene. So unless all the copies of a gene are inactivated, there will remain functional copies that will mask the effect of the mutation. Application of the VIGS technique led to the identification of critical roles for the ET- signaling pathway, as well as PAMP-triggered Immunity (PTI) in wheat�s resistance to FHB. The proven FHB-VIGS assay will also be an important tool to functionally assess the roles of new candidate genes identified by RNA-Seq studies. In addition to identifying important components of the FHB resistance pathways the researchers in this team collaborated with other scientists to identify genes involved in resistance to other pathogens and pests of wheat. These collaborations revealed genes involved in resistance of all grass species, including wheat, rice maize, barley and sorghum, to Cochliobolus carbonum, as well as wheat�s resistance to leaf rust, Pyrenophora tritici-repentis and the Russian wheat aphid. Accomplishments 01 Resistance to Fusarium head blight (FHB) is activated. We demonstrated that ethylene plays a critical role in activating resistance to FHB. Experiments in which wheat plants were genetically or chemically prevent from reacting to ethylene drastically reduced resistance, while studies that increased ethylene-signaling, improved resistance. The discovery th ethylene signaling makes a significant contribution to FHB resistance ha a major benefit to area research as many components in this pathway are already known and clear hypotheses for improving resistance can be teste As a part of this, we have generated transgenic wheat plants that have elevated expression of genes normally activated by ethylene, and once adequate seed is available they will be tested to see if they have improved resistance to FHB.
Impacts
(N/A)
Publications
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) 1. Identify plant genes whose pattern of expression changes in resistant compared to susceptible cereals when inoculated with viral or fungal pathogens. The research emphasis will be on barley and cereal yellow dwarf viruses, Fusarium head blight (scab), and leaf rust. 2. Develop a virus-induced gene silencing system (VIGS) as a tool to determine gene function in cereal crops. 3. Use the VIGs system to determine which cereal genes identified are essential in resistance and susceptibility responses (resistance pathway components). 4. Develop useful DNA markers for improvement of viral and fungal resistance. a. Develop markers for the genes verified to encode resistance pathway components in for use in Marker-Assisted Selection (MAS). b. Generate and characterize SSR-enriched libraries from oat. c. Identify DNA markers linked to the wheatgrass-derived Bdv3 and FHB resistance for MAS in wheat. Approach (from AD-416) Barley and cereal yellow dwarf viruses (YDV) are causing significant losses to small grain production. Fungal pathogens, such as Fusarium head blight and cereal rusts continue to cause major damage and losses. Development of genetic resistance in small grains to these pathogens is hindered by the lack of information about mechanisms that confer host- plant resistance and the lack of linked DNA markers to assist in the development of improved germplasm. Our approach will be to use new and existing molecular genetic methods such as gene expression arrays, transient gene silencing and to develop an improved understanding of the genetic and biochemical mechanisms that can confer host-plant resistance disease resistance and utilize this information to develop new strategies for enhancing genetic resistance to cereal viral and fungal diseases. We are working to identify the genetic mechanism that provides resistance to Fusarium head blight in wheat. Our work employs a novel virus-induced gene silencing (VIGS) assay to inactivate chosen wheat genes so that their role in FHB resistance can be tested. This approach has demonstrated that genes that promote ethylene-induced signaling are critical for FHB resistance. The VIGS tests have been confirmed independently through the use of chemicals that prevent ethylene- signaling. Chemicals that block ethylene-signaling also abolish resistance. More importantly, chemicals that increase ethylene-signaling result in increased resistance to FHB, strongly suggesting that our efforts to genetically engineer increased expression of ethylene signaling components will improve resistance. Accomplishments 01 Discovery of a critical role for ethylene signaling in FHB resistance. have made a significant discovery about the regulation of resistance to Fusarium head blight (FHB). We demonstrated that ethylene plays a critical role in activating resistance to FHB. Experiments in which whe plants were genetically or chemically prevented from reacting to ethylen drastically reduced resistance, while studies that increased ethylene- signaling, improved resistance. We have generated transgenic wheat plan that have elevated expression of genes normally activated by ethylene, a they will soon be tested to see if they have improved resistance to FHB. 02 Development of a rapid detection assay for Yellow dwarf viruses (YDV). PCR-based assay to rapidly diagnosing any of the five strains from different virus isolates across the country was developed. The test involves two steps. The first detects whether any of these viruses are present in a single 2 hr real-time quantitative PCR run. If any YDV is detected, a second run with a specific set of primers is able to distinguish them in between Barley-YDV or Cereal-YDV. The method has proven useful and to a wide range of diagnostic labs. It permits them t give the answers back to their farmers within days. Knowing in advance about the viruses in the fields will provide a better way to control the infection in the future for better yield.
Impacts
(N/A)
Publications
- Van Eck, L., Schultz, T., Leach, J.E., Scofield, S.R., Peairs, F.B., Botha, A., Lapitan, N.V. 2010. Virus-induced gene silencing of WRKY53 and an inducible phenylalanine ammonia-lyase in wheat reduces aphid resistance. Plant Biotechnology Journal. 9:1023-1032.
- Manning, V.A., Chu, A.L., Scofield, S.R., Ciufetti, L.M. 2010. Intracellular expression of a host-selective toxin, ToxA, in diverse plants phenocopies silencing of a ToxA-interacting protein, ToxABP1. New Phytologist. 187:1034-1047.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) 1. Identify plant genes whose pattern of expression changes in resistant compared to susceptible cereals when inoculated with viral or fungal pathogens. The research emphasis will be on barley and cereal yellow dwarf viruses, Fusarium head blight (scab), and leaf rust. 2. Develop a virus-induced gene silencing system (VIGS) as a tool to determine gene function in cereal crops. 3. Use the VIGs system to determine which cereal genes identified are essential in resistance and susceptibility responses (resistance pathway components). 4. Develop useful DNA markers for improvement of viral and fungal resistance. a. Develop markers for the genes verified to encode resistance pathway components in for use in Marker-Assisted Selection (MAS). b. Generate and characterize SSR-enriched libraries from oat. c. Identify DNA markers linked to the wheatgrass-derived Bdv3 and FHB resistance for MAS in wheat. Approach (from AD-416) Barley and cereal yellow dwarf viruses (YDV) are causing significant losses to small grain production. Fungal pathogens, such as Fusarium head blight and cereal rusts continue to cause major damage and losses. Development of genetic resistance in small grains to these pathogens is hindered by the lack of information about mechanisms that confer host- plant resistance and the lack of linked DNA markers to assist in the development of improved germplasm. Our approach will be to use new and existing molecular genetic methods such as gene expression arrays, transient gene silencing and to develop an improved understanding of the genetic and biochemical mechanisms that can confer host-plant resistance disease resistance and utilize this information to develop new strategies for enhancing genetic resistance to cereal viral and fungal diseases. Fusarium head blight (FHB) is major problem for wheat growers in the United States, Canada, Europe and China. Infection by the fungus, Fusarium graminearum, greatly reduces the yield and quality of grain. Unfortunately, the currently known sources of genetic resistance provide only partial protection against FHB. For this reason, a major focus of our work has been the identification of genes making significant contributions to FHB resistance in wheat (Objective 3). Gaining this knowledge should allow us to engineer improved FHB resistance. Our primary approach to this objective has been to silence candidate genes in normally FHB resistant wheat plants using a virus-induced gene silencing (VIGS) system (Objective 2). The VIGS studies provide strong indications for the function of genes in the FHB resistance pathway when it is observed that normally FHB resistant plants are susceptible after silencing the candidate gene. In the past year we have identified several new wheat genes as playing significant roles in FHB resistance. Additionally, in some cases we have been able to employ means other than VIGS to independently confirm the VIGS results. We are now beginning to generate transgenic wheat plants that will over express our most promising candidate genes. These will then be tested for improved FHB resistance. In collaborative studies, this VIGS approach has also identified genes required for aphid resistance. Barley and Cereal Yellow Dwarf Viruses continue to be a significant pathogen in small grain cereal growing regions in the USA. Using existing technologies and new DNA and RNA sequencing methods we have been examining and identifying genes whose expression patterns change when resistant and susceptible wheat and oat lines are inoculated with these viruses. A subset of these genes are being characterized to determine their role in the resistance response. Oat improvement has been hampered by a lack of molecular markers spread across the genome. In collaborative work with other scientists we have identified well over 5,000 DNA markers using different but complimentary approaches. While further work is needed to place these markers in specific locations within the oat genome they will provide a significant resource to oat breeders in the USA and globally. A virus detection method confirms that Barley and Cereal Yellow Dwarf Viruses are the most prevalent viruses in the Eastern US. This method has also shown that Wheat Streak Mosaic Virus also is present in 10-20% of the samples tested indicating that resistance to this virus needs to be integrated into soft- red winter wheat varieties. Accomplishments 01 Virus Epidemiology. ARS researchers at West Lafayette, Indiana have show from wheat plots at Vincennes, Indiana that yellow dwarf viruses specifically BYDV-MAV and BYDV-PAV continue to be the most prevalent viruses in winter wheat although the incidence declined from 75% in 2008 to 40% in 2009. During this same time period the incidence of wheat streak mosaic virus, previously thought to be a serious pathogen west of the Mississippi, increased from 30% to 50%. A regional analysis of wint wheat in North Carolina, Wisconsin, Georgia and Arkansas also demonstrat that BYDV-PAV and BYDV-MAV are the most prevalent viruses present. Thes studies showed that in all states 70 to 90% of all samples tested were infected with at least one virus, albeit some at a low level. These dat indicate that viruses continue to be a significant disease in wheat and other small grain cereals. This information is being used by plant breeders and plant pathologists to make decisions about incorporating virus disease resistance traits into improved germplasm and varieties. 02 Oat DNA Marker Development. The lack of sufficient oat DNA markers has made it impossible to use marker-assisted selection (MAS) in oat breedin Previous work developed approximately 2,500 markers. While this was a significant step forward, the size of oat genome requires additional markers to have full genome coverage. This is being accomplished with colleagues from North America by utilizing new DNA sequencing technology to identify single nucleotide polymorphisms (SNP). Preliminary work has identified approximately 5,000 SNPs from 20 different oat cultivars. These are currently being validated to identify those that are polymorph are spread across the genome, and can be used in oat marker-assisted- breeding for developing cultivars with improved quality traits and disea resistance. 03 Mechanism of resistance to Barley and Cereal Yellow Dwarf Virus. Wheatgrass-derived resistance to Barley and Cereal Yellow dwarf viruses wheat is proving to be quite effective but is not complete. Consequentl research to determine the mechanism of this resistance and how susceptib wheat responds to a virus infection is being done to identify genes that are involved in these processes. Recently, small RNAs that are less tha 40 nucleotides in length have been shown to regulate gene expression and have been shown to affect plant/virus interactions. In this study using high throughput sequencing, approximately over 120 million small RNAs we sequenced from wheat leaf tissue from resistant plants infected with Cereal Yellow Dwarf Virus. Regulatory RNAs found in all plant species have been identified from these data and are being characterized further However, the majority of small RNAs (60%) found, appear to be unique to wheat and the role they have in resistance and/or susceptibility is bein studied. 04 Fusarium head blight (FHB) resistance. The known genetic resistance in wheat and barley to the devastating fungal disease Fusarium head blight (FHB) is only partial. We are working to identify genes involved in FHB resistance so that we can engineer improved FHB resistance. In previous work we found that silencing several different genes known to act in a common signaling pathway all result in conversion to FHB susceptibility. To confirm the validity of the VIGS results we designed experiments to inactivate this pathway by a method unrelated to VIGS. These studies al resulted in the clear abrogation of FHB resistance and clearly add grea confidence to the initial findings. We are now in the process of generating transgenic plants that overexpress these genes so that we can test if this will confer increased levels of FHB resistance.
Impacts
(N/A)
Publications
- Scofield, S.R., Gillespie, M., Cakir, C. 2010. Rapid Determination of Gene Function by Virus-Induced Gene Silencing in Wheat and Barley. Crop Science. 50:77-84.
- Loutre, C., Wicker, T., Travella, S., Galli, P., Scofield, S.R., Fahima, T. , Feuillet, C., Keller, B. 2009. Two Genes Encoding Structurally Different CC-NB-LRR Proteins are Required for Lr10-Mediated Leaf Rust Resistance in Wheat of Two Ploidy Levels. Plant Journal. 60:1043-54.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) 1. Identify plant genes whose pattern of expression changes in resistant compared to susceptible cereals when inoculated with viral or fungal pathogens. The research emphasis will be on barley and cereal yellow dwarf viruses, Fusarium head blight (scab), and leaf rust. 2. Develop a virus-induced gene silencing system (VIGS) as a tool to determine gene function in cereal crops. 3. Use the VIGs system to determine which cereal genes identified are essential in resistance and susceptibility responses (resistance pathway components). 4. Develop useful DNA markers for improvement of viral and fungal resistance. a. Develop markers for the genes verified to encode resistance pathway components in for use in Marker-Assisted Selection (MAS). b. Generate and characterize SSR-enriched libraries from oat. c. Identify DNA markers linked to the wheatgrass-derived Bdv3 and FHB resistance for MAS in wheat. Approach (from AD-416) Barley and cereal yellow dwarf viruses (YDV) are causing significant losses to small grain production. Fungal pathogens, such as Fusarium head blight and cereal rusts continue to cause major damage and losses. Development of genetic resistance in small grains to these pathogens is hindered by the lack of information about mechanisms that confer host- plant resistance and the lack of linked DNA markers to assist in the development of improved germplasm. Our approach will be to use new and existing molecular genetic methods such as gene expression arrays, transient gene silencing and to develop an improved understanding of the genetic and biochemical mechanisms that can confer host-plant resistance disease resistance and utilize this information to develop new strategies for enhancing genetic resistance to cereal viral and fungal diseases. Significant Activities that Support Special Target Populations Studies to identify wheat genes contributing to resistance to Fusarium head blight (FHB) continues to be a major research focus. The genetic resistance to FHB currently available to breeders is partial and acts to limit the spread of the fungus rather than prevent infection. Nonetheless, it offers significant efficacy in controlling FHB. FHB resistance is not conditioned by major resistance genes, rather it appears to result from the cumulative effects of several quantitative trait loci (QTLs). At the beginning of these efforts no plant genes had been identified that play essential roles in resistance to this devastating fungal pathogen. The approach we are using is to use virus- induced gene silencing to silence wheat genes, which are candidates for being involved in FHB resistance, in a genotype that is normally resistant to FHB. These plants are then challenged with Fusarium and observed to see if they remain resistant or develop FHB. If VIGS causes the plants to become susceptible we have strong evidence that the candidate has a significant role in FHB resistance. The candidates that we are screening are chosen because they have been shown to be differentially expressed during interactions with Fusarium in wheat and barley. Through this VIGS screening we have now identified several genes that make significant contributions to FHB resistance. Efforts have continued to attempt to improve Barley Stripe Mosaic Virus (BSMV) as a tool for virus-induced gene silencing (VIGS). Much of this work in FY09 dealt with investigating the function of the BSMV gene Gamma b, which encodes a suppressor of silencing. We have compared wild type and mutant versions of the virus in which Gamma b has been mutated or completely deleted to see if this alters the area of the plant in which gene silencing occurs. This analysis has indicated that Gamma b is essential for combating the host silencing system and thereby permitting the spread of VIGS. BSMV derivatives lacking Gamma b are rapidly overcome by the silencing system and no further spread of VIGS occurs. Using a previously developed virus detection method, studies have shown that across a wide geographical area from Wisconsin through Georgia that Barley and Cereal Yellow Dwarf Viruses are the most predominant viruses affecting wheat and that mixed infections of these viruses occur in over 50% of the nearly 800 samples tested. The virus detection method has been improved by adding High Plains Virus and Triticum Mosaic Virus to make it more useful for use in the Great Plains region where these viruses are causing significant yield losses. This detection method can now simultaneously detect 10 different small grain cereal viruses. Technology Transfer Number of New/Active MTAs(providing only): 21
Impacts
(N/A)
Publications
- Held, M.A., Penning, B., Kessans, S.A., Yong, W., Scofield, S.R., Brandt, A.S., Carpita, N.C. 2008. Small-Interfering RNAs from Natural Antisense Transcripts Derived from a Cellulose Synthase Gene Modulate Cell Wall Biosynthesis in Barley. Proceedings of the National Academy of Sciences. 105:20534-10539.
- Scofield, S.R., Cakir, C. 2008. Evaluating the Ability of the Barley Stripe Mosaic Virus-Induced Gene Silencing System to Simultaneously Silence Two Wheat Genes. Cereal Research Communications. 36:217-222.
- Scofield, S.R., Nelson, R. 2009. Resources for Virus-Induced Gene Silencing (VIGS) in the Grasses. Plant Physiology. 149:152-157.
- Mudge, K., Goldschmidt, E., Scofield, S.R., Janick, J. 2009. A History of Grafting. Horticultural Reviews, Plant Breeding Reviews. 35:437-487.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) 1. Identify plant genes whose pattern of expression changes in resistant compared to susceptible cereals when inoculated with viral or fungal pathogens. The research emphasis will be on barley and cereal yellow dwarf viruses, Fusarium head blight (scab), and leaf rust. 2. Develop a virus-induced gene silencing system (VIGS) as a tool to determine gene function in cereal crops. 3. Use the VIGs system to determine which cereal genes identified are essential in resistance and susceptibility responses (resistance pathway components). 4. Develop useful DNA markers for improvement of viral and fungal resistance. a. Develop markers for the genes verified to encode resistance pathway components in for use in Marker-Assisted Selection (MAS). b. Generate and characterize SSR-enriched libraries from oat. c. Identify DNA markers linked to the wheatgrass-derived Bdv3 and FHB resistance for MAS in wheat. Approach (from AD-416) Barley and cereal yellow dwarf viruses (YDV) are causing significant losses to small grain production. Fungal pathogens, such as Fusarium head blight and cereal rusts continue to cause major damage and losses. Development of genetic resistance in small grains to these pathogens is hindered by the lack of information about mechanisms that confer host- plant resistance and the lack of linked DNA markers to assist in the development of improved germplasm. Our approach will be to use new and existing molecular genetic methods such as gene expression arrays, transient gene silencing and to develop an improved understanding of the genetic and biochemical mechanisms that can confer host-plant resistance disease resistance and utilize this information to develop new strategies for enhancing genetic resistance to cereal viral and fungal diseases. Significant Activities that Support Special Target Populations The milestones in this project are divided into three basic objectives with the long term goal of understanding the genetic basis for disease resistance in small grain crops to develop more disease resistant varieties. A) characterizing the response in wheat to infection by viral and fungal pathogens, B) identification of genes that may be involved in conferring resistance to fungal and viral pathogens, and C) identification of DNA markers that can be used to enhance the breeding for improved wheat and oat germplasm. Substantial progress has been achieved in the initial phase to enhance our knowledge of the response in small grain crops to important fungal and viral pathogens. Preliminary analyses have shown that a number of genes are both induced and repressed in wheat and oat lines infected with Barley and Cereal Yellow Dwarf Viruses. Research is ongoing to determine if these genes are directly or indirectly involved in resistance to these economically important viruses. Recently we developed a rapid method for detecting as many as eight different viruses in small grain crops. This is the only method capable of studying the epidemiology of cereal viruses within and across cereal growing regions in the US and worldwide. Results thus far have shown that Barley and Cereal Yellow Dwarf Viruses (YDV) are the most common viruses and that most infected plants usually contain two or more viruses. In addition to YDVs, Soilborne Wheat Mosaic Virus and Wheat Spindle Streak Mosaic Virus also are present in a significant number of samples from North Carolina, Indiana, and Missouri. This information is being used by plant breeders and plant pathologists to make decisions about incorporating virus disease resistance traits into improved germplasm and varieties. This method is now being used by plant pathogen diagnostic labs in six different states. Substantial progress has been achieved in developing DNA markers for use in wheat and especially oat. A library of over 1,000 potentially polymorphic oat markers have been identified and are currently being analyzed to determine their usefulness in generating genetic marker maps and for utility in breeding applications. With an international group of oat researchers and Diversity Array Technology, Pty Ltd, a high throughput molecular marker platform (Oat DArT) for Oat has been developed that contains over 2,000 new markers that will be available for use by oat researchers in 2009. The combination of SSR markers and Oat DaRT array molecular markers provides a significant new resource for oat improvement. This research progress is aligned with National Program 303 Plant Diseases, Component 1. Disease Diagnosis: Detection, Identification and Characterization of Plant Pathogens and Component Three - Plant Disease Resistance Problem Statement 3A Mechanisms of Plant Disease Resistance and 3B Disease resistance in new germplasm and varieties. Technology Transfer Number of New/Active MTAs(providing only): 3
Impacts
(N/A)
Publications
- Kong, L., Ohm, H.W., Anderson, J.M. 2007. Expression analysis of defense- related genes in wheat in response to infection by Fusarium graminearum. Genome. 50:1038-1048.
- Mahua, D., Anderson, J.M. 2008. Development of a multiplexed PCR detection method for Barley and Cereal Yellow Dwarf viruses, Wheat Spindle Streak Virus, Wheat Streak Mosaic Virus and Soil-Borne Wheat Mosaic Virus. Journal of Virological Methods. 148:17-24.
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