A high percentage of core effectors contribute to virulence of U. maydis

Previous comparative genomic studies have shown that many secreted effector proteins without known domains i.e., novel, are conserved only in the Ustilaginaceae family. By analyzing the secretome of 11 species within Ustilaginaceae, we identified 53 core homologous groups commonly present in this lineage. By collecting existing mutants and generating additional ones via conventional gene replacement or CRISPR-Cas9-based disruptions or deletions, we gathered 44 U. maydis strains lacking single core effectors as well as 9 strains containing multiple deletions of core effector gene families. Pathogenicity assays revealed that 20 of these 53 mutant strains were affected in virulence. Among the 33 mutants that had no obvious phenotypic changes, 13 carried additional, sequence-divergent, structurally similar paralogs of the deleted core effectors. This might explain why we failed to detect a virulence defect in these 13 mutants. Our study has identified seven uncharacterized single core effectors and one effector family contributing to virulence. Our approach to focus on core effectors efficiently allowed to prioritize effectors for further characterization (Schuster et al., 2024) and points to a new way to select effectors for functional studies also in other systems.