DNA methylation activates retron Ec86 filaments for antiphage defense

Highlights•Phage-encoded DNA cytosine methyltransferase (Dcm) is a trigger of the retron Ec86 system•Dcm methylates the stem-loop region of msDNA to activate retron Ec86•Cryo-EM analysis reveals the Ec86-effector filaments containing NAM and ADPr•The Ec86-effector filament is capable of hydrolyzing NAD(P)+SummaryRetrons are a class of multigene antiphage defense systems typically consisting of a retron reverse transcriptase, a non-coding RNA, and a cognate effector. Although triggers for several retron systems have been discovered recently, the complete mechanism by which these systems detect invading phages and mediate defense remains unclear. Here, we focus on the retron Ec86 defense system, elucidating its modes of activation and mechanisms of action. We identified a phage-encoded DNA cytosine methyltransferase (Dcm) as a trigger of the Ec86 system and demonstrated that Ec86 is activated upon multicopy single-stranded DNA (msDNA) methylation. We further elucidated the structure of a tripartite retron Ec86-effector filament assembly that is primed for activation by Dcm and capable of hydrolyzing nicotinamide adenine dinucleotide (NAD+). These findings provide insights into the retron Ec86 defense mechanism and underscore an emerging theme of antiphage defense through supramolecular complex assemblies.Graphical abstract