Structurally related phage helicases trigger type III-A3 retron-mediated anti-phage defense across diverse tailed phage families.

Retrons are tripartite bacterial systems composed of non-coding RNA (ncRNA), reverse transcriptase (RT), and effector proteins with diverse enzymatic domains. Here, we characterized Retron-Eco11, a type III-A3 retron associated with a phosphoribosyltransferase-like effector, and demonstrated that it mediates anti-phage defense and can be harnessed for genome editing. All three components-ncRNA, RT, and effector-are essential for defense. Retron-Eco11 protects Escherichia coli against multiple phages and is specifically activated by structurally related, phage-encoded helicases, including UvsW and D10 from phages T4 and T5. We show that these helicases trigger effector-mediated toxicity, leading to abortive infection, which is associated with phosphoribosyl pyrophosphate depletion due to PRTase activation, thereby altering nucleotide metabolism. UvsW interacts directly with multicopy single-stranded DNA, and effector protein activation requires the catalytic activity of phage-encoded helicases. Our data indicate that activation occurs without detectable effector release or measurable changes in the composition of the Retron complex. Beyond its defensive role, Retron-Eco11 enables targeted genome editing in bacterial and eukaryotic cells, underscoring the biological relevance and biotechnological potential of type III-A3 retrons.