2006 Annual Report 4d.Progress report. This project report serves to document research conducted under an assistance-type cooperative agreement between ARS and Cornell University. 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. 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.