Genome-wide atlas of lncRNAs in Mucorales reveals conserved regulators essential for fungal viability and development

Long non-coding RNAs (lncRNAs) are key regulators across eukaryotes, yet their functions in non-Dikarya fungal pathogens remain largely unexplored. Here, we provide a comprehensive identification and characterization of lncRNAs in the non-Dikarya fungal order Mucorales, a WHO high-priority group of opportunistic human pathogens. Using Mucor lusitanicus and the clinically relevant pathogen Rhizopus microsporus as models, we show that NDF lncRNAs exhibit conserved features, dynamic regulation during host interactions, and integration into gene regulatory networks. Although preferentially encoded in inactive chromatin compartments, lncRNA expression in R. microsporus is associated with N6-methyladenine (6mA). We further show that lncRNAs are targeted by canonical and non-canonical RNA interference (RNAi) pathways. Comparative genomics analyses identified conserved lncRNAs, including two essentials for viability (lncRNA2 and lncRNA4). Disruption of lncRNA4, even in heterokaryosis, severely impairs growth and filamentation. These results establish lncRNAs as important regulators of fungal physiology and pathogenicity, highlighting their potential as novel antifungal targets.