Improving resistance of traditional Italian tomato cultivars against biotic stress

This thesis begins with a general introduction and aims of the research as Section 1 which underscores the importance of traditional tomato varieties, such as the San Marzano cultivar, in agriculture. It highlights the vulnerabilities of these cultivars to various bacterial and fungal pathogens, which lead to significant crop losses. The introduction also explores the potential of gene-editing technologies, particularly CRISPR/Cas9, in addressing these challenges by enhancing disease resistance while preserving the desired traits of traditional cultivars. In Section 2, the focus is on the design of three knockout plasmids targeting the susceptibility genes SlPelo, SlDMR6-1, and SlMlo1. These genes were selected for their roles in weakening the plant’s defense mechanisms against pathogens. The CRISPR/Cas9-based constructs were assembled using the GoldenBraid [GB] cloning system and introduced into the San Marzano cultivar via Agrobacterium tumefaciens. The aim was to generate plants with improved resistance to biotic stress by disrupting these susceptibility genes. Section 3 covers the pathogenicity assays performed on tomato cultivars to assess their responses to bacterial and fungal pathogens. The assays included tests on PL mutant Micro-Tom plants, which carry a CRISPR-induced mutation in the SlPL gene. These experiments demonstrated how the targeted gene knockouts affected the plants' resistance, providing valuable insights into the effectiveness of CRISPR-mediated gene editing in combating plant diseases. Section 4 investigates the role of CsLOB1 and SlLOB1 in cell wall degradation during Xanthomonas citri infection. The study demonstrates that ectopic expression of these transcription factors in Nicotiana benthamiana and Micro-Tom tomato fruit leads to significant cell wall degradation and increased oligogalacturonide [OG] production. This release of OGs provides carbon sources for the pathogen, facilitating bacterial growth in the plant apoplast. The results confirm that LOB1 transcription factors play a key role in modulating host cell wall dynamics, enhancing the susceptibility of plants to Xanthomonas infections, and offering insights into potential strategies for mitigating such vulnerabilities. The thesis concludes by demonstrating the potential of CRISPR/Cas9 technology in improving disease resistance in traditional Italian tomato cultivars, offering a promising pathway for developing resilient crops without compromising their agricultural value.