The Pleiades are a cluster of fungal effectors that inhibit host defenses

Biotrophic plant pathogens secrete effector proteins to manipulate the host physiology. Effectors suppress defenses and induce an environment favorable to disease development. Sequence-based prediction of effector function is impeded by their rapid evolution rate. In the maize pathogen Ustilago maydis, effector-coding genes frequently organize in clusters. Here we describe the functional characterization of the pleiades, a cluster of ten effector genes, by analyzing the micro- and macroscopic phenotype of the cluster deletion and expressing these proteins in planta. Deletion of the pleiades leads to strongly impaired virulence and accumulation of reactive oxygen species (ROS) in infected tissue. Eight of the Pleiades suppress the production of ROS upon perception of pathogen associated molecular patterns (PAMPs). Although functionally redundant, the Pleiades target different host components. The paralogs Taygeta1 and Merope1 suppress ROS production in either the cytoplasm or nucleus, respectively. Merope1 targets and promotes the auto-ubiquitination activity of RFI2, a conserved family of E3 ligases that regulates the production of PAMP-triggered ROS burst in plants. Author summary The genomes of filamentous plant pathogens encode for hundreds of protein-coding effector genes. These secreted proteins target different host components to modify its physiology and promote disease. Effector coding genes usually evolve fast and lack largely functional domains, making the prediction of their function a difficult task. In the maize pathogen Ustilago maydis, effector coding genes usually cluster together in the genome. Within clusters, new genes are thought to emerge by duplication events followed by quick selection. Here we study the functional relevance of one of these clusters. In the pleiades, 8 out of 10 genes function to suppress host defenses, irrespective of their sequence relationship, which constitutes the first functional elucidation of an effector cluster in U. maydis. Additionally, we detect the hallmarks of a neo-functionalization event for two paralogs within the cluster of which one effector targets the host proteasomal degradation pathway in the nucleus and the other acts in the plant cytoplasm or the plasmamembrane. Our study shows the functional relevance of effector gene organization and highlights the redundancy, yet mechanistic diversity, in effector functions.