cDNA-guided functional selection uncovers selective defense systems against RNA phages

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. IMPORTANCEBacteria harbor a broad repertoire of antiphage defense systems, but our understanding of mechanisms that target RNA phages remains limited, being heavily biased toward defenses against DNA phages. By applying a cDNA-based functional selection strategy, this study overcomes a major obstacle in defense-gene discovery and uncovers previously uncharacterized genes that represent bona fide components of the bacterial immune arsenal against RNA phages. The identification and characterization of Zowangsin (ZwsA), a NERD-domain RNA endonuclease that selectively cleaves specific signatures within RNA phage genomes, establish targeted RNA degradation as a central principle of bacterial defense against RNA phages. More broadly, this work expands the conceptual framework of bacterial antiviral immunity and illustrates the utility of functional selection for uncovering cryptic immune systems with implications for phage biology, RNA biology, and biotechnology. HIGHLIGTHSO_LIcDNA-based functional screen identifies six defense systems that restrict RNA phages C_LIO_LIThese defense systems are enriched within genomic islands of Pseudomonas aeruginosa C_LIO_LIZws, Szs, and Mws systems confer selective defense against RNA phages C_LIO_LIZwsA is a signature-selective RNA endonuclease that targets phage genomic RNA C_LI