Cryo-EM structure analysis of phage ΦXacm4-11 that infects the phytopathogen <i>Xanthomonas citri</i>

ABSTRACT Very few bacteriophages that infect Xanthomonas species have been characterized genetically and only one 3D structure, the capsid of a siphovirus that infects the phytopathogen Xanthomonas citri , has been determined at high resolution. This study presents the annotated DNA sequence and detailed structural analysis of ΦXacm4-11, a podovirus that infects Xanthomonas citri , shedding light on its unique architecture and functional attributes, providing insights into the molecular mechanisms underlying host recognition and infection. Annotation of the genome revealed conserved features among related phages, but also distinct genetic elements that may contribute to ΦXacm4-11’s specificity toward X. citri. Genes associated with host recognition and infection were identified, including the genes potentially coding for the receptor-binding proteins (RBPs) at the tail fibre tip, offering insights into their role in bacterial attachment. Using high-resolution cryo-electron microscopy, we resolved the architecture of the mature, pre-released virion, revealing a T7-like head-tail assembly with a well-defined portal-tail complex embedded at a unique fivefold vertex. Our findings provide a detailed view of the structural and functional components of ΦXacm4-11, furthering our understanding of its molecular interactions with X. citri and its potential application in phage therapy against phytopathogens. SIGNIFICANCE STATEMENT Bacteriophages are increasingly recognized as powerful tools to control bacterial pathogens in medicine and agriculture, yet the structural basis of host recognition and genome delivery remains poorly understood for most phages. Here, we present a comprehensive structural and functional analysis of ΦXacm4-11, a podovirus that infects the plant pathogen Xanthomonas citri . By combining genome annotation, proteomics, and high-resolution cryo-electron microscopy, we reveal the complete architecture of the mature virion and its specialized portal-tail machinery. Our results show how this short-tailed phage deploys an internal injection device to penetrate the bacterial cell envelope and highlight structural features linked to type IV pilus-dependent infection. These findings provide insights into phage entry mechanisms and establish ΦXacm4-11 as a model for engineering biocontrol strategies.