Villares Portugal, R. | Search bioRxiv

bioRxiv2026

Cryo-EM structure analysis of phage {Phi}Xacm4-11 that infects the phytopathogen Xanthomonas citri

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 {Phi}Xacm4-11, 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 {Phi}Xacm4-11s 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 {Phi}Xacm4-11, furthering our understanding of its molecular interactions with X. citri and its potential application in phage therapy against phytopathogens. SIGNIFICANCE STATEMENTBacteriophages 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 {Phi}Xacm4-11, 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 {Phi}Xacm4-11 as a model for engineering biocontrol strategies.

biochemistry↗

bioRxiv2024

Protein-RNA condensation kinetics via filamentous nanoclusters

Protein-RNA phase separation is at the center of membraneless biomolecular condensates governing cell physiology and pathology. Using an archetypical viral protein-RNA condensation model, we determined the sequence of events that starts with sub-second formation of a protomer with two RNAs per protein dimer. Association of additional RNA molecules to weaker secondary binding sites in this protomer kickstarts crystallization-like assembly of a molecular condensate. Primary nucleation is faster than the sum of secondary nucleation and growth, which is a multistep process. Protein-RNA nuclei grow over hundreds of seconds into filaments and subsequently into nanoclusters with circa 600 nm diameter. Cryoelectron microscopy reveals an internal structure formed by incoming layers of protein-RNA filaments made of ribonucleoprotein oligomers, reminiscent of genome packing of a nucleocapsid. These nanoclusters progress to liquid condensate droplets that undergo further partial coalescence to yield typical hydrogel-like protein-RNA coacervates that may represent the scaffold of large viral factory condensates in infected cells. Our integrated experimental kinetic investigation exposes rate limiting steps and structures along a key biological multistep pathway present across life kingdoms.

biophysics↗