Amoroso, Ciro Gianmaria (2022) GENOMIC APPROACHES TO IMPROVE TOMATO RESPONSE TO MULTIPLE STRESS. [Tesi di dottorato]

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Climate changes are reducing cultivable areas and available natural resources, affecting food security in many countries. In this light, producing more food for a growing population represent a critical challenge for humanity in the coming decades. Tomato (Solanum lycopersicum) is among the most widespread horticultural crop and is commonly used as a model plant for genetic studies due to its short biological cycle, the availability of genetic and genomic resources and the recently fully sequenced genome. Globally, many biotic and abiotic stresses adversely affect tomato growth, production and quality, inducing tremendous economic and yield losses. The introduction of R-genes in cultivated tomatoes could enhance resistance to different stresses. However, this process is tedious and time-consuming and results in high costs over the long term. Therefore, developing novel strategies to obtain tomato cultivars with enhanced resilence to multiple stress conditions is critical for plant scientists. This work was aimed to develop tomato cultivars resistant to multiple stresses using innovative genome editing approaches. As a starting point, ten tomato cultivars of interest for the company were evaluated for their phenotypical traits, the presence or absence of molecular markers associated with resistance loci, and the in vitro regeneration rates. The regeneration step is crucial for producing stable genomic-transformed plants. Phenotypical and molecular analysis comparisons allowed to select SanMarzano2 as the most interesting line for the genetic improvement program, due to its strategic importance for the company, the lack of genetic resistances, and the good number of regenerated shoots. Successively, we conducted a large-scale literature analysis, exploring dozens of trancriptomic studies, to identify suitable target genes for the CRISPR/Cas9 editing. Twelve studies were selected, and the raw transcriptomic data of tomato plants exposed to different biotic and abiotic stress were retrieved and analyzed de novo using the bioinformatic platform A.I.R. DEGs induced by each stress were compared for the identification of genes involved in response to different stress. Data cross-comparison allowed the identification of several pathways activated in response to fungi, bacteria, virus, pest, and different abiotic stress such as drought, salinity, cold and oxidative stress. In particular, pathogen recognition, signaling, hormone metabolism, transcription, defensive proteins, and other important cell compounds resulted perturbed after stress recognition. One-hundred and twenty-six genes were identified to be involved in 5 response to different biotic and abiotic stresses. This list was used as a query against several tomato-stress studies reported in the literature. Interestingly, forty-nine genes have also been reported in other tomatoes-stress interactions, while seventy-seven DEGs have never been characterized in tomato before. These genes could be used for further investigations using genetic engineering techniques for assessing their role in plant multiple stress response. In addition, a bibliographic search with specific keywords allowed the identification of twenty-seven genes involved in tomato resistance or susceptibility to vascular pathogens or multiple stress responses. The cross-examination of comparative transcriptomic data and bibliographical research allowed the selection of WATI and HyPRPI as optimal gene targets for the genome editing experiment. To this scope, the CRISPR/Cas9 system was used to produce deleterious deletions on the two genes in order to increase tomato resistance to vascular pathogens and abiotic stress. A large number of SanMarzano2 and MoneyMaker genotypes edited plants were obtained. Transformed plants were checked at the molecular level and transferred in vivo to obtain the next selfing generations and to assess basic phenotypic traits. The gene knockout produced slwatI plants with dwarf and stunted phenotypes compared with slhyprpI plants, while the double mutated plants slwatI:slhyprpI showed a tall intermediate phenotype. Further analyses will be conducted to test tolerance to biotic and abiotic stress.

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