Phage homing endonuclease facilitates evasion of the antiphage defenses through segmental amplification of anti-defense factors

Bacteriophages deploy diverse genetic strategies to overcome bacterial immunity, yet the mechanisms that enable immune escape remain incompletely understood. Here, we show that the phage homing endonuclease SegB facilitates immune evasion by facilitating segmental amplification of anti-defense genes. The anti-phage defense module Septu protects host bacteria against phage T6 by cleaving the variable loop of tRNATyr to inhibit phage replication. Although T6 encodes its own tRNATyr gene, this alone is insufficient to compensate for the loss caused by Septu-mediated cleavage. We found that SegB, encoded adjacent to the tRNATyr gene in the T6 genome, enables immune evasion by amplifying a genomic segment containing the full-length tRNATyr. This repeat expansion increases tRNATyr expression, allowing the phage to escape Septu immunity. Notably, SegB also facilitates the amplification of anti-defense genes that counteract the distinct defense module OLD in trans. These findings suggest that SegB-mediated segmental amplification represents a general strategy by which phages can circumvent diverse bacterial immune systems.