Dual recognition of phage SSB triggers DRT4 for antiphage defense
Defense-associated reverse transcriptase 4 (DRT4) protects bacteria against phages through single-stranded DNA (ssDNA) synthesis and activation of an effector RNase. How precisely phage recognition is coupled to RNase activation remains unclear. Here, we combine structural, biochemical, and genetic analyses to define the requirements for DRT4 activation. Cryo-electron microscopy structures capture DNA synthesis and provide a structural basis for preferential purine incorporation. Screening against the BASEL phage collection reveals protection against diverse phages and identifies the Bas17-encoded ssDNA-binding (SSB) protein gp44 as a DRT4 trigger. We show that DRT4 requires both recognition of its ssDNA product and direct protein-protein interactions of gp44 for RNase activation. Together, our findings support a previously unrecognized "two-factor authentication" model in which ssDNA-mediated recruitment of a phage SSB cooperates with specific protein interactions to license abortive infection for antiphage defense.