Activation of the Pathogen-Inducible Gst1 Promoter of Potato after Elicitation by Venturia inaequalis and Erwinia amylovora in Transgenic Apple ( Malus × Domestica )

Rather than using a constitutive promoter to drive transgenes for resistance against fungal and bacterial diseases in genetic engineering of apple (Malus × domestica) cultivars, a promoter induced only after infection was preferred. The ability of the Pgst1 promoter from potato (Solanum tuberosum L.) to drive expression of the gusA reporter gene was determined in two genotypes of apple: the fruit cultivar Royal Gala and the M.26 rootstock. β-glucuronidase activity in the transgenic lines grown in a growth chamber was determined quantitatively using fluorometric assays and compared to the activity in Cauliflower Mosaic Virus (CaMV) 35S promoter-driven transgenic lines. In both apple genotypes, the Pgst1 promoter exhibited a low level of expression after bacterial and fungal inoculation compared to the level obtained with the PCaMV35S promoter (15% and 8% respectively). The Pgst1 promoter was systematically activated in apple at the site of infection with a fungal pathogen. It was also activated after treatment with salicylic acid, but not after wounding. Taken together, these data show that, although the Pgst1 promoter is less active than the PCaMV35S promoter in apple, its pathogen responsiveness could be useful in driving the expression of transgenes to promote bacterial and fungal disease resistanc

Performance and long-term stability of the barley hordothionin gene in multiple transgenic apple lines

Introduction of sustainable scab resistance in elite apple cultivars is of high importance for apple cultivation when aiming at reducing the use of chemical crop protectants. Genetic modification (GM) allows the rapid introduction of resistance genes directly into high quality apple cultivars. Resistance genes can be derived from apple itself but genetic modification also opens up the possibility to use other, non-host resistance genes. A prerequisite for application is the long-term performance and stability of the gene annex trait in the field. For this study, we produced and selected a series of transgenic apple lines of two cultivars, i.e. ‘Elstar’ and ‘Gala’ in which the barley hordothionin gene (hth) was introduced. After multiplication, the GM hth-lines, non-GM susceptible and resistant controls and GM non-hth controls were planted in a random block design in a field trial in 40 replicates. Scab resistance was monitored after artificial inoculation (first year) and after natural infection (subsequent years). After the trial period, the level of expression of the hth gene was checked by quantitative RT-PCR. Four of the six GM hth apple lines proved to be significantly less susceptible to apple scab and this trait was found to be stable for the entire 4-year period. Hth expression at the mRNA level was also stable

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 bligh

HIPM is a susceptibility gene of Malus: reduced expression reduces susceptibility to Erwinia amylovora

Fire blight, a devastating disease caused by the bacterium Erwinia amylovora, is a major threat to apple crop production. To improve our understanding of the fire blight disease, and to identify potential strategies to control the pathogen, we studied the apple protein “HrpN-interacting protein from Malus” (HIPM), which has previously been identified as interacting with the E. amylovora effector protein HrpN. Transgenic apple plants were generated with reduced HIPM expression using an RNAi construct, and were subsequently analysed for susceptibility to E. amylovora infection. Lines exhibiting a greater than 50% silencing of HIPM expression showed a significant decrease in susceptibility to E. amylovora infection. Indeed, a correlation between HIPM expression and E. amylovora infection was identified, demonstrating the crucial role of HIPM during fire blight disease progression. Furthermore, an apple oxygen-evolving protein (MdOEE) was identified via a yeast two hybrid screen to interact with HIPM. This result was confirmed with bimolecular fluorescence complementation assays, and leads to new hypotheses concerning the response mechanism of the plant to E. amylovora, as well as the mechanism of infection of the bacterium. These results suggest that MdOEE and particularly HIPM are promising targets for further investigations towards the genetic improvement of appl