To determine the function of candidate fire blight resistance genes, RNAi mutants of M. 26 apple rootstock were produced using an efficient multiplex transformation system. Five RNAi EST-silencing vectors were used in each transformation experiment to allow selection of up to five types of mutants from a single experiment. RNAi-silencing constructs were created using ESTs associated with response of apple to E. amylovora that were selected based upon transcript profiling data and bioinformatics. Twenty candidate genes in six functional categories were evaluated by using the pHELLSGATE8 RNAi-mediated gene silencing vector to silence specific EST derived sequences and then observing the resulting resistance phenotype. Silencing of candidates genes is confirmed by RT-qPCR. Mutants are phenotyped following inoculation of young plants with E. amylovora by determining area under the disease progress curve (AUDPC), cumulative percent of current season’s shoot length blighted and the population of E. amylovora in young plants determined by qPCR. Silencing of some candidates had no observable resistance phenotype. Sequence derived from EST GenBank DR999015 resulted in the significant down-regulation of two hypoxia induced protein genes and a significant reduction of AUDPC in comparison to parent M.26.