RNAi mutants for determination of candidate gene function in resistance of apple to Erwinia amylovora (Fire blight)

To determine the function of candidate apple genes that confer resistance to fire blight, 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 Erwinia amylovora that were selected based upon transcript profiling data and bioinformatics. Candidate genes in six functional categories were evaluated by using the pHELLSGATE8 RNAi-mediated gene silencing vector to silence a specific EST derived sequence and then observing the resulting resistance phenotype. Silencing of candidate genes was confirmed by RT-qPCR. Transgenic lines were phenotyped following inoculation of young plants with E. amylovora by determining the area under the disease progress curve (AUDPC), the cumulative percentage of shoot length that was blighted and the population of E. amylovora in young plants as determined by qPCR. Some of transgenic lines with the p8L01 and pfbox silencing construct had a significantly lower population of E. amylovora than non-transformed M.26. The disease severity in the lines with the p4H09 silencing construct was significantly different from that of nontransformed M.26. Also some lines with the pfbox, p8L01, pPO53 and pSGT1 silencing constructs had significantly lower disease severity than non-transformed M.26. The genes associated with reduced disease-severity and/or lower E. amylovora population are potentially useful in maker assisted breeding and GE for cultivars and rootstocks with increased resistance to fire blight