A recombinant hybrid provides insights into gene regulation, pathogenesis, and tumorigenesis of phytopathogenic smut fungi.

The closely related maize smut fungi Ustilago maydis and Sporisorium reilianum share genome structures but differ in their pathogenic behavior. U. maydis causes local tumors, while S. reilianum spreads systemically and affects inflorescences. To investigate the genetic basis of these differences, we generate an interspecific recombinant hybrid (recombinant U. maydis x S. reilianum hybrid [rUSH]) carrying the S. reilianum mating type. rUSH exhibits in planta proliferation and an S. reilianum-like phenotype, except for teliospore formation. Transcriptome profiling reveals that pathogenicity-related effector orthologs are induced in rUSH but not in a wild-type hybrid control. Comparative transcriptomics identifies 253 differentially expressed one-to-one effector orthologs with cis-, trans-, and a rUSH-specific phenomenon of cis/trans. CRISPR-Cas9 uncovers three novel virulence factors among the rUSH-specific expressed effectors. Ultimately, we pinpoint the transcription factor UmHdp2 as a key regulator of U. maydis-induced tumorigenesis. Our findings highlight the utility of a recombinant, interspecific hybrid in unraveling the molecular mechanisms underlying pathogenic differences in closely related fungal pathogens.