Strain-Specific Infection of Phage AP1 to Rice Bacterial Brown Stripe Pathogen Acidovorax oryzae

Bacteriophage (phage) AP1 has been reported to effectively lyse Acidovorax oryzae, the causative agent of bacterial brown stripe in rice. However, phage AP1 exhibits strain-specific lysis patterns. In order to enhance the potential of phages for biological control of rice bacterial brown stripe, this study investigated the possible mechanism of strain-specific infection by characterizing phage AP1 and its susceptible (RS-2) and resistant (RS-1) strains. Based on the current classification standards and available database information, phage AP1 was classified into the class Caudoviricetes, and it is a kind of podophage. Comparative analysis of the susceptible and resistant strains showed no significant differences in growth kinetics, motility, biofilm formation, or effector Hcp production. Interestingly, the resistant strain demonstrated enhanced virulence compared to the susceptible strain. Prokaryotic expression studies indicated that six putative structural proteins of phage AP1 exhibited varying degrees of binding affinity (1.90-9.15%) to lipopolysaccharide (LPS). However, pull-down assays and bacterial two-hybrid analyses revealed that only gp66 can interact with four host proteins, which were identified as glycosyltransferase, RcnB, ClpB, and ImpB through immunoprecipitation and mass spectrometry analyses. The role of LPS in the specific infection mechanism of phage AP1 was further elucidated through the construction of knockout mutant strains and complementary strains targeting a unique gene cluster (wbzB, wbzC, wbzE, and wbzF) involved in LPS precursor biosynthesis. These findings provide novel insights into the mechanisms of phage-host specificity, which are crucial for the effective application of phage AP1 in controlling rice bacterial brown stripe.