Leaderless RiPPs expand the repertoire of fungal secondary metabolites

Authors: PARK, SUNG CHUL - University Of Wisconsin; OSTER, LIVIA, D.S. - University Of Minnesota; GOLAN, JACOB - Collaborator; MYERS, JILLIAN - University Of Michigan; PRINGLE, ANNE - University Of Wisconsin; Drott, Milton; KELLER, NANCY - University Of Wisconsin

Submitted to: Proceedings of the National Academy of Sciences (PNAS)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/15/2026
Publication Date: 2/11/2026
Citation: Park, S., Oster, L., Golan, J., Myers, J., Pringle, A., Drott, M.T., Keller, N.P. 2026. Leaderless RiPPs expand the repertoire of fungal secondary metabolites. Proceedings of the National Academy of Sciences (PNAS). 123(7). Article e2529748123. https://doi.org/10.1073/pnas.2529748123.
DOI: https://doi.org/10.1073/pnas.2529748123

Interpretive Summary: Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a class of chemical produced by bacteria, fungi, animals and plants. These chemicals are often toxic, and contribute to defense or pathogenesis of the organisms that produce them. RiPP compounds in fungi have always been found to include a 'leader' sequence that is separated from the 'core' sequence during the maturation of the final compound. Here we identify the first fungal RiPP ever discovered without a leader sequence. We demonstrate that there are actually several different 'leaderless' RiPPs across fungal species. These leaderless RiPPs are found in both the native European and invasive U.S. populations of the death-cap mushroom, but the compounds are produced several order of magnitude more in the invasive range. Together our results offer a paradigm shift in our understanding of RiPP compounds across fungi and perhaps Eukaryotes, offering new opportunities to identify toxins in this class and understand RiPP-associated pathogenesis. Our results also suggest that modifications to canonical RiPP structure may be important in the success of invasive fungi in new environments, offering new opportunity to control the spread of introduced microorganisms.

Technical Abstract: Ribosomally synthesized and post-translationally modified peptides (RiPPs) are secondary metabolites produced by bacteria, fungi, animals, and plants. Fungal RiPP precursors canonically have a leader sequence that is cleaved during maturation, with conserved residues guiding processing. The first described fungal RiPPs were the MSDIN-derived peptides responsible for the lethality of the Deadly Amanita. In this study, we upend the conventional structure of MSDINs, discovering that a subclass of the MSDIN family has diversified among species without a leader sequence. This marks the first empirical example of a leaderless RiPP in fungi. We used a combinatorial analysis of NMR and MS/MS coupled with a bioinformatic pipeline to describe four peptides that are produced by leaderless MSDIN genes. We also characterized a mature MSDIN that is lacking in a proline residue at the end of the core sequence thought necessary for processing by the prolyl oligopeptidase, POPB. We investigated how these non-canonical genes might influence the invasive ecology of Amanita phalloides, a deadly fungus native to Europe and invading the U.S. West Coast. Leaderless and proline-less MSDIN transcripts were expressed several orders of magnitude higher than canonical MSDINs, with significantly higher expression in invasive populations. Our results offer a paradigm shift in our understanding of Eukaryotic RiPP architectures and suggest that differential regulation of non-canonical RiPPs may play a role in the invasion biology of the world's deadliest mushroom.