A Cryo-EM Portrait of a Phytopathogen-Infecting Phage

Bacteriophage research is flourishing due to their potential applications, but its historical focus on a small set of models left much of their diversity unexplored. The Xanthomonadaceae family is among the most important phytopathogens worldwide, making their associated phages particularly relevant. While many Xanthomonas phages have been isolated and characterized microbiologically, few genomes have been sequenced and structural information is scarce. Cryo-EM allows the obtention of high-resolution data, useful for understanding infection mechanisms and prospectively guiding strategies against bacteria-related plant diseases. Thus, we aim to determine the major architectural components of the ΦXacm4-11 virion using cryo-EM. ΦXacm4-11 was propagated on Xanthomonas citri strain 306. Adsorption and one-step growth assays were used to determine kinetic parameters of infection. The phage genome was sequenced by Illumina, assembled into a single contig and manually annotated. Virion proteins were identified by SDS-PAGE and LC-MS/MS. Cryo-EM data were collected on a Titan Krios microscope and processed using a framework in RELION to obtain asymmetric (C1) and symmetric-focused reconstructions (I, capsid; C6, tail). Atomic models were built using AlphaFold and refined in ChimeraX, Coot and Phenix. ΦXacm4-11 exhibits a latency of ~60 min and a burst size of ~299 particles per infected cell. The linear 43.4 kb genome encodes 63 ORFs organized into functional modules. Cryo-EM analysis revealed a T=7 laevo capsid composed of HK97-like major capsid proteins and cement proteins. A complex portal-adaptor-nozzle assembles at a unique fivefold vertex, together with internal densities consistent with a DNA-protein injection apparatus. High-resolution maps enabled detailed modeling of major tail components, while the tail fiber region displayed substantial structural heterogeneity. This study provides the first structural characterization of the Xanthomonas-infecting podovirus ΦXacm4-11, that may serve as a model to investigate structure-function relationships in T7-like phages and T4P-dependent infection. These insights also support the rational development of Xanthomonas phage-based biocontrol strategies.