Evolution of phage tail fiber proteins to counter bacterial resistance and improve biocontrol efficacy in plant disease models.

Phage therapy is being used to combat pathogenic bacterial infections that threaten plant, animal, and human health. However, its application remains limited by high host specificity and the emergence of bacterial resistance. In this study, we addressed the key issues in phage therapy using rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) strain N1 and its lytic phage NP1. Strain N1 acquired resistance to the phage NP1 through mutations and downregulation of lipopolysaccharide (LPS) biosynthesis genes. A directed evolution assay using phage NP1 and the resistant strain N1R resulted in the development of phage E12-2, which overcame bacterial resistance, expanded its host range and improved bacterial suppression by targeting alternative LPS binding sites. Moreover, genome analysis identified two amino acid substitutions (V303L and G317V) in its tail fiber protein. Additionally, phage E12-2 improved disease control efficiency by 51 % compared to the wild-type phage NP1 and induced plant immunity in a plant disease model. These findings enhance our understanding of how bacteria-phage evolution shapes the dynamics of phage therapy in plants.