Reverse transcribed ssDNA derepresses translation of a retron antiviral protein

Retrons are bacterial immune systems that prevent phage propagation by initiating a toxic response within infected hosts. Previously characterized retrons produce abundant multicopy single-stranded DNA (msDNA) by reverse transcription, which acts as an antitoxin in the absence of phage. However, we describe here a non-canonical mechanism for type VI retrons, which do not produce detectable msDNA in the absence of phage yet still provide defense. Focusing on Retron-Vpa2 from V. parahaemolyticus, we show broad defense against phages and identify triggers of the system when expressed in E. coli. Within the retron operon, we find a highly enriched, structured transcript that we term a hybrid RNA (hyRNA). The hyRNA contains the reverse transcription template and a toxin coding sequence that is repressed by a riboregulator. In our model of triggering, phage infection induces accumulation of msDNA, which hybridizes to the riboregulator and enables RNase H cleavage, derepressing translation of the toxin.