Improving fire blight resistance in susceptible apple cultivars by different biotechnological approaches

Fire blight, caused by the bacterium Erwinia amylovora (E. amylovora), is one of the most economically important apple (Malus x domestica) pathogens worldwide. Various chemical and biological approaches can be applied to deal with the disease, but none of these is decisive. Such strategies are also prohibited in many countries due to their potential impact on human health and environment. To date, the most efficient strategy for controlling E. amylovora is thus to breed resistant/tolerant apple cultivars by manipulating one or multiple plant genes, which are associated with resistance or susceptibility to the disease. Within this context, classical breeding or genetic engineering can be applied. While conventional breeding is still considered a time-consuming and laborious process, genetic engineering methodologies represent rapid, precise and powerful alternatives to insert the desired trait into the crop of interest. In this thesis, we exploit different biotechnological approaches on the one hand to improve fire blight resistance trait by knocking-out a known susceptibility gene and on the other hand to investigate potential disease-related key genes. At first, we develop a CRISPR/Cas9-FLP/FRT-based gene editing system, mediated by Agrobacterium tumefaciens, to knock-out the fire blight susceptibility gene MdDIPM4 and generate apple (‘Gala’ and ‘Golden Delicious’) cultivars with reduced susceptibility to the disease and a minimal trace of exogenous DNA. Several transgenic lines were screened by sequencing to identify mutations in MdDIPM4. An editing efficiency of 75% was observed. Candidate lines showing loss-of-function mutation were inoculated with E. amylovora and a significant reduction (of about 40%) in disease symptoms was observed compared to wild-type plants. No CRISPR/Cas9 off-targeting activity was detected in five potential off-target regions. Thus, with the aim of removing the ‘entire’ T-DNA in those lines with reduced susceptibility to the pathogen, the FLP/FRT system was induced and the excision of the T-DNA was validated. This work demonstrates for the first time the development and application of a CRISPR/Cas9-FLP/FRT-based editing system for the production of ‘clean’ fire blight resistant apple cultivars Secondly, we investigate the apple miRNA MdmiR285N which is predicted to play a key role in the post-transcriptional regulation of 35 RNA transcripts coding for different disease resistance proteins. A complex network of potential transcriptional regulatory elements involved in plant growth and development, and in response to different hormones and stress conditions has been identified in MdmiR285N promoter in both apple and the model plant species Arabidopsis thaliana. Moreover, Spatio-temporal expression of MdmiR285N has been assessed in plants at physiological growth conditions and in response to bacterial pathogens. Our results suggest that MdmiR285N is a multifunctional microRNA which may control different processes, such as biotic stress response, plant growth and development. In parallel, a methodological work has been carried out for a precise and rapid characterization of the transgenic apple lines produced. A quantitative, rapid and cost-effective method has been developed, based on real-time PCR to quantify the copy number of nptII marker gene in apple lines and to evaluate its elimination after the activation of the recombinase system. This method may be valuable for those institutions committed to tracing ‘gmo’ apple products.