Alternative isoforms and phase separation of Ref1 repress morphogenesis in Cryptococcus

Hyphal morphogenesis is a defining characteristic of sexual development in the basidiomycete fungus Cryptococcus neoformans. Sexual development affords Cryptococcus adaptability and resilience in response to environmental stressors and mediates immunogenicity in mammalian hosts. Znf2 is a transcriptional activator and the master regulator of the yeast-to-hypha transition. Despite precedent for negative regulation in eukaryotic development broadly and hyphal morphogenesis in particular, no negative regulators specific to sexual development in Cryptococcus have been identified. Through gene expression correlational analysis with ZNF2, we identified a basidiomycete-specific negative regulator of hyphal morphogenesis REF1 (CNAG_07982/CND00490). We demonstrated the ability of Ref1 to inhibit transcription of hyphal morphogenesis genes controlled by Znf2. We further established Ref1 as a negative regulator with functionally distinct isoforms that both block morphogenesis during asexual proliferation. The Ref1-Znf2 axis is critical for guiding Cryptococcus through the complex and dynamic morphological transition associated with sexual development. For all samples, strains were grown on filamentation-suppressing YPD medium in the dark. H99 background strains were grown at 30 degrees Celsius and XL280 background strains were grown at room temperature.