Genome-wide Discovery of lncRNAs in Mucorales Reveals Essential Roles in Development and Fungal Biology

Long non-coding RNAs (lncRNAs) emerged as key regulators across eukaryotes, yet their functions in early-diverging fungal (EDF) pathogens remain largely unknown. Here, we provide the first comprehensive identification and characterization of lncRNAs in the EDF order Mucorales, a threatening and WHO high-priority group of opportunistic human pathogens. In this work, we focus on the two major models of this group: Mucor lusitanicus and the clinically relevant pathogen Rhizopus microsporus . We show that EDF lncRNAs exhibit conserved features, dynamic regulation during host interactions, and integration within critical gene regulatory networks. Despite lncRNAs being preferentially encoded in inactive chromatin compartments, we found that their expression is associated with 6mA presence in R. microsporus . Additionally, we also found that lncRNAs can be targeted by both the canonical RNA interference pathway and the non-canonical RNA interference mechanism. Comparative genomics revealed a subset of evolutionarily conserved lncRNAs, including two essential for fungal viability (lncRNA2 and lncRNA4). LncRNA4 disruption, even in heterokaryosis, resulted in severely affected growth and filamentation. These results establish lncRNAs as indispensable regulators of fungal physiology and pathogenicity, highlighting their potential as novel antifungal targets.