Bae, H.-W.; Ki, H.-J.; Choi, S.-Y.; Cho, H.-G.; Woo, C.-H.; Kim, M.-J.; Chun, H.-J.; Cho, Y.-H.

Bacteria encode diverse antiphage defense systems, yet mechanisms that target RNA phages remain comparatively underexplored. Here, we used a cDNA-based functional selection strategy to systematically identify genes that confer resistance to RNA phage infection independently of receptor variation in Pseudomonas aeruginosa. This approach uncovered previously uncharacterized antiphage defense systems, most of which are located within genomic islands, consistent with their being bona fide components of bacterial immune systems. Several systems conferred selective resistance to RNA phages and their carriage was associated with pilin variability, suggesting layered anti-phage immunity. Among these systems, Zws is the most prevalent RNA phage defense system and functions as a multidomain effector. Structural modeling and in vitro cleavage assays showed that ZwsA is an RNA endonuclease that selectively cleaves RNA phage genomes through a predicted NERD domain. Together, these findings expand the current framework of bacterial antiphage immunity and highlight the power of functional genomics to uncover cryptic components of the bacterial antiviral arsenal.