2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Fruit trees, to be productive, require favorable environments, both biotic and abiotic. Among abiotic stresses, acute freeze damage can eliminate entire orchards, and drought stress is a continual and serious threat to U.S. agriculture that causes billion-dollar losses. Even trees that survive freezing temperatures or drought have reduced vigor, longevity, and productivity. As an example, it was estimated that in New York State alone, 25,000 apple trees were lost to winter damage in 2003-2004 at a value of 2.5 million dollars. Annual losses in the U.S. for all crops are estimated to be almost one billion dollars. Similarly, between 1978 and 1995, average crop losses due to drought in the U.S. exceeded $1.2 billion each year. Since 1995, the US has experienced 3 major droughts with losses estimated at over 1 billion dollars for each occurrence. Recent drought monitoring results suggest that the U.S. West Coast drought will continue to affect crops in the northwestern states. Among diseases, fire blight is easily transmitted, difficult to control, and can be catastrophic. A single fire blight epidemic that occurred in southwest Michigan in 2000 was estimated to have caused the death of 350,000 to 450,000 trees and the removal of 1,500 to 2,300 acres of apple orchards resulting in an economic loss of $42 million. Streptomycin is the primary control agent, but resistant strains of the pathogen, Erwinia amylovora, are appearing in some areas. Zinc deficiency is another serious issue in fruit production. Over 30 percent of the world’s soils are deficient in this essential micronutrient. Foliar applications are only partially effective and when repeated over time may lead to Zn soil contamination. Genomic analysis of herbaceous annual model plants, especially Arabidopsis, has to some degree elucidated the molecular basis of processes to overcome these problems. Genomic knowledge, including gene identification and function, is advancing in all the areas described above, i.e., environmental stress resistance, disease resistance, and micronutrient accumulation. How this body of knowledge applies to woody perennials, however, is unclear. Genomic research has been identified as a research priority in the Technology Roadmap for Tree Fruit Production because of its potential to enhance crop performance, improve food quality, and increase farm profits. Genetic improvement of apple and many other fruit trees by conventional breeding methods is very slow and difficult because of heterozygosity, long generation time, and self-incompatibility. As the research identifies and develops candidate genes for amelioration of stress injury, a technology must also be developed to introduce those genes into the tree genome. Biotechnology can be used to some degree to overcome the obstacles to breeding by introducing genes directly into current commercial cultivars to add the needed traits. There is considerable market resistance to genetically engineered fruit; if the technology is to be useful, "clean" methods of genetic transformation need to be developed. With grafted trees, the possibility exists of developing genetically engineered rootstocks with needed traits, while maintaining the non-engineered status of the scions (and the harvested fruit). This strategy needs to be developed and its feasibility tested. This research has the potential to provide economic benefits to growers as a direct result from reduced losses to cold weather, drought, and disease; disease resistant germplasm will result in fewer chemical inputs to control diseases; growers and consumers will benefit from a more reliable supply of high quality fruit, less dependent on weather conditions, thus, prices will be more stable over the season. Increased water use efficiency will enhance tree productivity and lower production costs under both normal and dry conditions, including orchards that are irrigated, as well as those that are not. Engineered rootstocks will bring the benefits of recombinant DNA technology to fruit trees, without resulting in a genetically engineered product to sell in a market reluctant to accept them. The research outlined in this project falls within NP 302 – Plant Molecular and Biological Processes and mainly addresses Component 2B - Plant Interactions with Their Environment. The research also addresses Component 1B: Applying Genomics to Crop Improvement and Component 3A: Improving and Assessing Genetic Engineering Technology. The objectives are relevant to ARS research goals, because they will identify genes in temperate tree fruits that are responsible for resistance to disease and weather losses (ARS Strategic Plan Performance Measure 1.2.7) and will provide producers and other customers with scientific information and technology that increases production efficiency, safeguards the environment, and reduces production risks and product losses (ARS Strategic Plan Performance Measure 1.2.5). 2.List by year the currently approved milestones (indicators of research progress) FY 2007 Objective 1 Hypothesis 1 – Specific genes or sets of genes are expressed in response to environmental stress and determine the degree of resistance. 1. Complete identification of low temperature/short day (LT/SD) genes and proteins in peach bark tissues. Hypothesis 2 – Specific genes or sets of genes are expressed in response to fire blight challenge and determine the degree of resistance. 2. Complete identification of ESTs (gene fragments) associated with early (24 h) and late (48 h) fire blight infection events. Objective 2 Hypothesis 1 – Gene flow and expression in transgenic apple can be more precisely controlled. 3. Complete Northern and Southern analyses of apple rootstock lines overexpressing Zn-transporter genes. 4. Complete construction of transformation vectors that uses a flower-specific promoter. FY 2008 Objective 1 Hypothesis 1 – Specific genes or sets of genes are expressed in response to environmental stress and determine the degree of resistance. 1. Complete creation of transgenic apple lines over-expressing or silencing the cold binding transcription factor (CBF). 2. Complete sequencing of water-deficit responsive genes in apple. Hypothesis 2 – Specific genes or sets of genes are expressed in response to fire blight challenge and determine the degree of resistance. 3. Identify genes associated with resistance and susceptibility to fire blight. Objective 2 Hypothesis 1 – Gene flow and expression in transgenic apple can be more precisely controlled. 4. Complete qualitative evaluation of the ability of GUS/RNAi-silencing in rootstocks to suppress GUS expression in scion. 5. Complete evaluation of the response of apple rootstock lines overexpressing a zinc transporter gene to sub- and supra-optimal zinc levels. 6. Complete construction of transformation vector that utilizes a flower-specific promoter to express a dehydrin gene. 7. Obtain transgenic Arabidopsis plants that utilize a flower-specific promoter to express a GUS gene (GUS genes are used to assess the effectiveness of the gene promoter). FY 2009 Objective 1 Hypothesis 1 – Specific genes or sets of genes are expressed in response to environmental stress and determine the degree of resistance. 1. Identification of LT/SD induced genes in apple and their expression patterns. Hypothesis 2 – Specific genes or sets of genes are expressed in response to fire blight challenge and determine the degree of resistance. 2. Complete identification of ESTs and potential candidate genes associated with resistance and susceptibility to fire blight. Objective 2 Hypothesis 1 – Gene flow and expression in transgenic apple can be more precisely controlled. 3. Complete evaluation of silencing of carbohydrate metabolism genes on suppression of carbohydrate metabolism genes in scion. 4. Complete evaluation of ZNT lines (apple lines overexpressing zinc transporter genes) to sub-and supra-optimal levels of zinc. 5. Complete evaluation of Arabidopsis lines using a flower-specific promoter to express the GUS marker gene. 6. Obtain transgenic lines of Arabidopsis that utilize a flower-specific promoter to express a dehydrin gene for increased stress resistance. FY 2010 Objective 1 Hypothesis 1 – Specific genes or sets of genes are expressed in response to environmental stress and determine the degree of resistance. 1. Complete evaluation of the role of CBF in cold acclimation of apple. 2. Complete identification of LT/SD –induced genes in apple. Hypothesis 2 – Specific genes or sets of genes are expressed in response to fire blight challenge and determine the degree of resistance. 3. Complete identification of ESTs associated with non-host resistance and identification of potential candidate genes for enhanced resistance. 4. Validate expression of candidate genes in resistant and susceptible cultivars. Objective 2 Hypothesis 1 – Gene flow and expression in transgenic apple can be more precisely controlled. 5. Complete identification of transgenic apple rootstock lines overexpressing Zn- transporter genes suitable for further field evaluation. 6. Complete evaluation of Arabidopsis lines that utilize a flower-specific promoter to express a dehydrin gene for increased cold tolerance. FY2011 Objective 1 Hypothesis 1 – Specific genes or sets of genes are expressed in response to environmental stress and determine the degree of resistance. 1. Integrate data on environmental stress induced gene expression in fruit crops into current knowledge of gene networks and response pathways. Hypothesis 2 – Specific genes or sets of genes are expressed in response to fire blight challenge and determine the degree of resistance. 2. Complete identification of ESTs associated with effector-dependent and effector-independent pathogenesis. 3. Integrate data on fire blight induced genes expression in apple into current knowledge of pathogen and stress-induced response networks. Objective 2 Hypothesis 1 – Gene flow and expression in transgenic apple can be more precisely controlled. 4. Determine if field evaluation of RNAi-silenced rootstocks for scion trait modification is justified. 5. Complete all studies utilizing transgenic apple lines overexpressing a zinc transporter gene. 6. Obtain transgenic apple lines that utilize a flower-specific promoter to express a dehydrin gene for increased freezing tolerance of flower buds. 4a.List the single most significant research accomplishment during FY 2006. Differential regulation of two peach dehydrin genes by low temperature and drought. The detrimental effects of environmental stress on the health and productivity of fruit trees result in significant economic losses to growers and higher prices for consumers. There is a need to identify native genes in fruit crops that confer resistance to adverse environmental conditions. Researchers at the USDA-ARS, Kearneysville, WV, have isolated two genes belonging to a family of stress-related genes (dehydrins) that are differentially regulated by either low temperature or drought. These results may be used to develop molecular markers and quality trait loci (QTLs) for environmental stress in fruit crops. Within NP 302: Plant Molecular and Biological Processes, this research addresses Component 2B: Plant Interactions with Their Environment. Addresses CRIS Objective 1 (Hypothesis. 1)– Specific genes or sets of genes are expressed in response to environmental stress and determine the degree of resistance. 4b.List other significant research accomplishment(s), if any. Characterization of the Temporal Response of Apple to Fire Blight Disease. Fire blight, caused by the bacterium Erwinia amylovora, is a destructive disease of apple, pear, and other plants in the rose family (Rosaceae). Researchers at the USDA, ARS, Kearneysville, WV, and Pennsylvania State University, York, PA, have used cDNA suppressive-subtractive hybridization to characterize the genomic temporal response of apple to fire blight infection. The response of apple to the fire blight pathogen was far more rapid than previously demonstrated and indicates an active interaction between host and pathogen resulting in a rapid response cascade. These results will be used to identify the most meaningful assay times in future studies aimed at characterizing the response of apple to fire blight disease and at identifying critical genes controlling resistance. Within NP 302: Plant Molecular and Biological Processes, this research addresses Component 2B: Plant Interactions with Their Environment. Addresses CRIS Objective 1 (Hypothesis. 2)– Specific genes or sets of genes are expressed in apple in response to fire blight challenge and determine the degree of resistance. 4c.List significant activities that support special target populations. None. 4d.Progress report. This report serves to document research conducted under a reimbursable agreement between ARS and the Washington State Tree Fruit Research Commission (1931-21000-016-01T). Additional details can be found in the report for the parent project 1931-21000-016-00D, "Using Functional and Applied Genomics to Improve Stress and Disease Resistance in Fruit Trees." The project was terminated in July, 2004. USDA-ARS provided expertise to produce transgenic apple plants (cv Gala) overexpressing a cytosolic ascorbate peroxidase (APX) gene. Resulting lines exhibited increased resistance to oxidative stress induced by freezing, high temperature, and UV-B. Industry supplied general expertise and partial support. This report serves to document research conducted under a trust agreement between ARS and the Washington Tree Fruit Research Commission (1931-21000-016-02T). Additional details can be found in the report for the parent project 1931-21000-016-00D, "Using Functional and Applied Genomics to Improve Stress and Disease Resistance in Fruit Trees." The goal of the project is to develop technology to modify traits in scion, or fruiting, varieties through genetically engineered rootstocks. ARS scientists at Kearneysville, WV, constructed plant transformation vectors, and selected transgenic apple scions and rootstocks to investigate graft-transmissible gene silencing across graft-union junctions in apple. These included vectors to silence the expression of critical enzymes in sorbitol synthesis in apple, vectors to express the beta-glucuronidase (GUS) marker gene in apple, and vectors to subsequently silence the GUS marker gene. The advantage of using the GUS marker for these experiments is that it is relatively easy to monitor and quantify, allowing sampling throughout the tree and over time to determine uniformity of silencing in the scion. The GUS marker and the sorbitol silencing constructs were transformed into ‘Royal Gala’ scion, and the GUS and sorbitol silencing constructs were transformed into M.26 rootstock. Transgenic plants are currently being characterized and the appropriate silenced clones will be selected for the research. To determine if genetically engineered rootstock can modify scion traits by gene silencing, 'Royal Gala' that was genetically engineered to produce GUS ('Royal Gala+GUS') will be grafted to M.26 rootstock engineered to silence GUS (M.26-GUS). By determining the amount of GUS expression in 'Royal Gala+GUS' trees grafted onto M.26-GUS rootstock in comparison to 'Royal Gala+GUS' trees grafted onto M.26 rootstock we will determine if the engineered rootstock is capable of silencing GUS in the scion and if there is uniform silencing throughout the entire tree and over time. To determine if a genetically engineered rootstock will be as effective in altering a trait as a genetically engineered scion, 'Royal Gala' will be grafted to M.26 rootstock engineered to silence sorbitol production (M.26-sorbitol). In addition, 'Royal Gala' and 'Royal Gala' silenced for sorbitol production ('Royal Gala-sorbitol') will be grafted to M.26 rootstock. By comparing the level of sorbitol in 'Royal Gala-sorbitol' trees with the sorbitol level of 'Royal Gala' trees grafted to M.26 and M.26-sorbitol rootstocks we will determine if the silenced rootstock is as effective in reducing sorbitol production as the silenced scion. If the genetically engineered rootstocks are successful in altering the GUS marker and sorbitol traits in the scion, this technology would be used to improve commercially important traits, such as increased productivity, fruit quality and pest resistance. The application of rootstock mediate trait modification could overcome many of the hurdles facing transgenic fruits, including greatly reduced environmental concerns since transgenic pollen would not be produced by the transgenic rootstock, improved consumer acceptance since fruit would not be transgenic, and greater ease of commercialization since a single transgenic rootstock could be used to enhance the value of many different commercial fruiting varieties. This project report serves to document research conducted under a reimbursable agreement between ARS, The Pennsylvania State University, Cornell University and the USDA-CSREES National Research Initiative (1931-21000-016-03R). Additional details can be found in the report for the parent project 1931-21000-016-00D, "Using Functional and Applied Genomics to Improve Stress and Disease Resistance in Fruit Trees". Fire blight, caused by the bacterium Erwinia amylovora, is a destructive disease of apple, pear and other plants in the rose family (Rosaceae). ARS scientists at Kearneysville, WV, working in collaboration with ARS scientists at Geneva, NY, scientists at Cornell University, Geneva, NY and scientists at The Pennsylvania State University, York, PA are using a functional genomic analysis to characterize the response of apple to fire blight disease. Fire blight-susceptible (M.26) and -resistant (G.41) apple rootstocks were challenged with E. amylovora and the transcriptome of the two cultivars is being characterized by cDNA subtractive/PCR-suppressive hybridization and cDNA-AFLP. In addition, bioinformatic approaches were used to identify publicly available apple ESTs uniquely associated with E. amylovora infected apple or similar to Pseudomonas syringae pv. tomato infected Arabidopsis ESTs. Gene silencing is being used to elucidate the role of specific candidate genes in resistance and susceptibility. Two types of RNAi plant transformation vectors designed to facilitate the evaluation of large EST populations are being evaluated:. 1)those that use GATEWAY technology to facilitate the rapid generation of hairpin RNA-encoding constructs and. 2)those that use a 3’ untranslated region-inverted repeat to enhance sense-RNAi. The genomics research undertaken in this project will elucidate the poorly understood mechanisms responsible for the resistance and susceptibility of apple to fire blight and identify molecular markers for useful genes. Marker assisted selection could then be used by plant breeders to efficiently select superior apple varieties with improved resistance to fire blight disease. This project report serves to document research conducted under an assistance-type cooperative agreement between ARS and The Pennsylvania State University (1931-21000-016-04G). Additional details can be found in the report for the parent project 1931-21000-016-00D, "Using Functional and Applied Genomics to Improve Stress and Disease Resistance in Fruit Trees". Fire blight, caused by the bacterium Erwinia amylovora, is a destructive disease of apple, pear and other plants in the rose family (Rosaceae). The overall goal of this project is to use a functional genomic analysis to characterize the response of apple to fire blight disease and, in so doing, identify new opportunities for improving fire blight resistance in apple. Dr. Robert Farrell, Jr., at The Pennsylvania State University, York, PA working in collaboration with ARS scientists at Kearneysville, WV, is identifying genes that are turned on (up-regulated) and turned off (down-regulated) in apple within the first few hours following challenge with the fire blight pathogen and those that respond two- to three days after challenge. All organisms respond to changes in their environment and other forms of stress, including infection by bacteria and viruses, by switching certain genes on while simultaneously switching other genes off. Among the several state-of-the-art methods currently in use to study changes in gene expression, the method known as subtractive suppression PCR is perhaps the most inclusive global method for studying the effects of exposure to fire blight. In this approach, genes that are expressed in common between two different plants are subtracted away using a differential hybridization method, rendering those genes which are uniquely expressed under one set of circumstances. To date, this approach has been used to compare gene expression in infected versus uninfected apple, in resistant versus susceptible apple, and at various time points after exposure to the fire blight pathogen in order to study the temporal progression of the disease. The up- and down-regulated genes have been cloned and are currently being analyzed to identify those associated with the resistance and susceptibility of apple to fire blight disease. The genomics research undertaken in this project will elucidate the poorly understood mechanisms responsible for the resistance and susceptibility of apple to fire blight and identify molecular markers for useful genes. Marker assisted selection could then be used by plant breeders to efficiently select superior apple varieties with improved resistance to fire blight disease. In addition, the project has supported two very talented Penn State seniors and provided them an opportunity to participate in and contribute to the research project. This project report serves to document research conducted under an assistance-type cooperative agreement between ARS and Cornell University (1931-21000-016-05G). Additional details can be found in the report for the parent project 1931-21000-016-00 D, "Using Functional and Applied Genomics to Improve Stress and Disease Resistance in Fruit Trees". Fire blight, caused by the bacterium Erwinia amylovora, is a destructive disease of apple, pear and other plants in the rose family (Rosaceae). The overall goal of this project is to use a functional genomic analysis to characterize the response of apple to fire blight disease and, in so doing, identify new opportunities for improving fire blight resistance. Dr. Herb Aldwinckle at Cornell University, Geneva, NY, working in collaboration with ARS scientists at Kearneysville, WV, is studying changes in gene expression in fire blight-susceptible (M.26) and -resistant (G.41) apple rootstocks following challenge with the fire blight pathogen by the method known as cDNA amplified fragment length polymorphism (cDNA-AFLP). In addition, RNA interference (RNAi) is being used to silence specific candidate genes and thereby, elucidate their role in resistance and susceptibility. To develop a high-throughput method for generating RNAi mutants in apple, M.26 rootstock will be transformed with a mixture of several RNAi vectors to allow the selection of several types of mutants from a single transformation experiment. Initially, M.26 was transformed with three single vectors and a mixture of the three vectors to determine if the use of mixed vector inoculum adversely affects transformation frequency. The transformation frequency was not adversely affected by the use of mixed vector inoculum. Individual transgenics are currently being analyzed to determine if they contain single or multiple RNAi insertions. Multiple RNAi insertions would complicate the determination of gene function, and therefore the transformation conditions that minimize the occurrence of multiple insertions will be determined. The genomics research undertaken in this project will elucidate the poorly understood mechanisms responsible for the resistance and susceptibility of apple to fire blight and identify molecular markers for useful genes. Marker assisted selection could then be used by plant breeders to efficiently select superior apple varieties with improved resistance to fire blight disease. 5.Describe the major accomplishments to date and their predicted or actual impact. None. This is a new CRIS. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? None. This is a new CRIS. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). "Addressing Public Concerns About GMOs", presented to the annual meeting of the West Virginia Academy of Sciences, Shepherd University, April 22, 2006. "Subtractive/Suppressive Hybridization Analysis of Genes Regulated by Low Temperature and Short Photoperiod in Peach Bark", presented to the Plant and Microbial Adaptation to Cold conference, Salsomaggiore Terme, IT, May 23-27, 2006.