Functional Analysis of Spinach Downy Mildew Effectors

Abstract

Plants are susceptible to microbial infections that can cause disease and severe yield losses. Although plant–microbe interactions have been extensively studied in model systems, translating this knowledge into improved disease resistance in crops remains challenging, especially for non-model pathosystems with limited biological understanding and experimental tools. One such system is spinach downy mildew, caused by the obligate biotrophic oomycete Peronospora effusa (P. effusa). This rapidly evolving pathogen readily overcomes newly deployed resistance traits in spinach, highlighting the need for novel and more durable resistance. A deeper understanding of the P. effusa–spinach interaction may provide new leads to support the development of new pest management strategies. In chapter 2, we investigated how P. effusa deploys its gene repertoire during infection. Using time-resolved transcriptomic profiling across the complete asexual infection cycle, we show that P. effusa undergoes extensive and coordinated gene expression reprogramming associated with major developmental transitions. These changes involve stress responses, signalling pathways, metabolism, macromolecule biosynthesis, and distinct waves of effector expression. Several predicted virulence factors, including effectors, localise to genomic regions resembling pathogenicity islands and display coordinated expression patterns, suggesting that genome architecture contributes to the regulation of virulence. To enable functional studies in spinach, chapters 3 and 4 focus on establishing bacterial platforms for effector analysis in planta. In chapter 3, I evaluate Agrobacterium-mediated transient gene expression in spinach. Although reporter genes, including RUBY, could be expressed, expression levels were low and variable, and the absence of cell death responses indicated that further optimisation is required before this system can be used for resistance gene discovery. In chapter 4, I explored 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 a suitable chassis for effector screening in spinach. Using this system, I assessed the effects of candidate effectors on disease symptoms, bacterial proliferation, and reactive oxygen species production, establishing the first deployable platform for functional effector studies in spinach. Together, this thesis advances understanding of P. effusa infection and provides both candidate virulence genes and experimental tools for functional studies, thereby supporting targeted and durable resistance breeding in spinach.