Identification and Functional Characterization of Candidate Effectors in the Wheat Pathogen Zymoseptoria tritici

Zymoseptoria tritici is a significant wheat pathogen responsible for Septoria tritici blotch (STB) disease and for causing up to 50% yield losses globally. Understanding how Z. tritici utilizes its effector repertoire to promote virulence is essential for developing effective resistance strategies. Despite the economic importance of this pathogen, only a few effectors have been functionally characterized, and genome-editing tools for Z. tritici remain limited, restricting functional studies of its virulence factors. This dissertation aims to: 1) describe different transcriptome profiles activated in Z. tritici during infection of susceptible, resistant, and non-host species, to identify candidate effector genes that are activated at early and transition to necrotrophic stages; 2) assess the role of candidate effectors in modulation of PTI and ETI responses through transient expression in N. benthamiana; 3) implement CRISPR-Cas9-medianted gene knockout for functional genomics of Z. tritici; and 4) generate a knockout strain for an effector gene implicated in modulation of host immunity. We identified crucial time points where Z. tritici dynamically regulates sets of genes. The primary differences in pathogen gene expression between susceptible and resistant interactions occur at the transition to the necrotrophic stage, around 10 days post-inoculation (dpi). At 3 dpi, Z. tritici activates a large set of genes in both susceptible and resistant interactions, but this response was reduced in the non-host interaction. Our findings suggest that early-stage transcriptional profiles are similar in resistant and susceptible wheat, diverging by 6 dpi, while host and non-host responses differ primarily during the early infection stages. We identified 32 effector candidates with high expression during early stages and the transition to the necrotrophic stage. Structural predictions and heterologous expression provided insights into their potential functions and subcellular localization. Functional characterization of seven effectors revealed that the presence of a signal peptide significantly influenced their activity on host defense responses. For instance, Mycgr3107904, reduced chitin-induced reactive oxygen species (ROS) production but induced necrosis when expressed with its signal peptide, suggesting a dual function at different infection stages. Conversely, Mycgr394290 increased ROS accumulation and triggered cell death when the signal peptide was included. Mycgr3109710 increased ROS accumulation when expressed without the signal peptide and attenuates ROS production when the signal peptide was included. To functionally validate these effectors, we applied CRISPR-Cas9 to disrupt five effector genes, marking the first successful implementation of this technology in Z. tritici. Two knockout mutants obtained for Mycgr3107904 exhibited delayed disease progression on susceptible wheat and distinct colony morphology in vitro, demonstrating its role in infection. This study establishes CRISPR-Cas9 as a viable tool for Z. tritici functional genomics. Collectively, by elucidating effector functions and developing genetic tools for Z. tritici, this work advances our understanding of fungal pathogenesis and provides a foundation for future disease management strategies in wheat.