Dual phage sensing activates an RNA-targeting reverse transcriptase for anti-phage defense

Defense-associated reverse transcriptases (DRTs) mediate diverse antiphage responses, yet how they sense phage infection and couple unconventional DNA synthesis to antiviral effector activation remains poorly understood. Here, we characterize DRT4 as a dual-sensor immune system that integrates two distinct phage-derived signals. Cryo-EM analysis reveals that DRT4 assembles into a dimer-of-trimers hexamer, with each protomer catalyzing protein-primed, template-independent synthesis of a covalently linked ssDNA product. DRT4 displays a strong preference for purine nucleotides, determined by base-specific interactions within the polymerase active site. Using phage escape genetics, we identify two independent activators of DRT4: the phage ribonucleotide reductase NrdAB, which increase intracellular dNTP levels, and the SSB-like protein 0087, which directly binds the DRT4-generated ssDNA. Activated DRT4 exhibits dNTP-dependent RNase activity, linking phage sensing to RNA cleavage and abortive infection. Together, our findings show DRT4-generated ssDNA as a signaling intermediate that integrates phage-induced metabolic changes with direct recognition of viral replication proteins, revealing how protein-primed DNA synthesis can be repurposed for infection sensing and antiviral effector activation.