Melanie Mendel

Just like humans and animals, plants are susceptible to microbial infections that can cause disease. In agricultural ecosystems that rely on genetically uniform plants grown at high intensity, such diseases can spread rapidly and lead to severe yield losses. Although the biological processes governing plant–microbe interactions have been extensively studied in model systems, translating these insights and research methods into improved disease resistance in crops remains challenging. This is particularly true for non-model pathosystems, where both biological understanding and experimental tools are often limited. One such poorly understood system is spinach downy mildew, caused by the obligate biotrophic oomycete P. effusa. P. effusa is a rapidly evolving pathogen that readily overcomes newly deployed resistance traits in spinach, posing a persistent challenge to sustainable crop protection. This dynamic highlights the need for novel and efficient resistance breeding. A deeper understanding of the P. effusa–spinach pathosystem is therefore essential to inform targeted and durable disease control. In recent years, genomic resources for both spinach and P. effusa have improved substantially. However, how the P. effusa gene repertoire is deployed over the course of infection to establish successful colonisation remains largely unknown. Moreover, experimental research into gene function in both host and pathogen is severely constrained by the lack of robust molecular toolboxes for functional genomics. This limitation, for instance, prevents the study of pathogen virulence factors, including effector proteins, which facilitate infection by manipulating host cellular processes and shaping the infection niche. Importantly, these effectors can also be recognised by the spinach immune system, triggering effective defence responses. Understanding effector deployment and function is therefore central to advancing resistance breeding. In chapter 2, this thesis investigates how the P. effusa gene repertoire is dynamically utilised during infection of susceptible spinach. Using time-resolved transcriptomic profiling across the complete asexual infection cycle, I show that P. effusa undergoes extensive and coordinated gene expression reprogramming associated with major developmental transitions. These changes encompass stress responses, signalling pathways, metabolic processes, macromolecule biosynthesis, and distinct waves of effector expression. Notably, multiple predicted virulence factors, including effectors, localise to specific genomic regions with features resembling pathogenicity islands and display coordinated expression patterns. These findings suggest that genome architecture contributes to the transcriptional regulation of virulence throughout the P. effusa infection cycle. While these insights provide a framework for prioritising candidate virulence genes, they also underscore the need for experimental systems to validate and explore gene function directly in spinach. Accordingly, chapters 3 and 4 focus on establishing and assessing heterologous biotechnological platforms for functional effector analysis in planta. In chapter 3, I evaluate Agrobacterium-mediated transient gene expression, a widely used system in model plants, for its applicability in spinach. By optimising experimental parameters, I achieved low but variable expression of reporter constructs, including RUBY. However, the absence of cell death upon expression of conserved cell death elicitors, despite responses to recombinant proteins, indicates that Agrobacterium-based effector screening in spinach requires further refinement for resistance gene discovery. In chapter 4, I therefore explore an alternative approach based on Type III Secretion System-mediated effector delivery. I identified Pseudomonas syringae pv. tomato DC3000 D36E, a strain lacking endogenous Type III effectors, as suitable for effector screening in spinach. Using this system, I functionally screened a set of effectors and assessed their impact on disease symptoms, bacterial proliferation, and suppression of reactive oxygen species production. This work establishes Type III Secretion System-mediated delivery as a first deployable platform for functional effector studies in spinach. Together, the findings of this thesis advance our understanding of the molecular mechanisms underpinning P. effusa infection and provide a foundation for prioritising candidate genes for functional studies, as well as for developing experimental platforms for effector analysis in spinach. This work therefore contributes to the knowledge base and methodological capacity required for targeted, knowledge-driven resistance breeding, ultimately supporting the development of more durable and sustainable disease control strategies in the P. effusa–spinach pathosystem.