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bioRxiv · 10.1101/2025.09.29.678632

Understanding the infection mechanism of {Phi}8 using infection method in Pseudomonas syringae pv. Phaseolicola strain: In-situ CryoET analysis

Kumar, S.·Robinson, C. F.·Bobe, D.·Khayat, R.
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Abstract

The Cystoviridae family strain {Phi}8 is an ideal model for studying the virus-host interactions and viral assembly mechanisms. In this study, we explored {Phi}8 infection kinetics in P. syringae pv. phaseolicola host cells using a combination of host lysis assays, transmission electron microscopy (TEM), cryo-focused ion beam (Cryo-FIB) milling, and in-situ cryo-electron tomography (Cryo-ET). Purified {Phi}8 was infected with host cells and observed at different time points. The host-pathogen infection was detected using TEM studies. The infected sample underwent Cryo-FIB milling. Our findings indicated that the infected host cell membrane changes its shape during infection. We were able to visualize the various stages of {Phi}8 assembly using subtomogram averaging. These stages included the unpackaged procapsid (PC) having a size of [~]41 nm and the double-stranded RNA (dsRNA)-packaged procapsid having a size of [~]54 nm. The packaged PC represented the most prevalent intermediate stages during viral assembly. Notably, the majority of complete virions isolated from the host cell were found outside the host cell, revealing the extracellular predominance of fully constructed particles. These findings emphasize the importance of stable intermediate stages in viral assembly and maturation of {Phi}8. This work advances our understanding of viral assembly and complex host-cell interactions. HighlightsO_LISuccessfully isolated and infected Pseudomonas syringae pv. phaseolicola (Pph) host cells with {Phi}8. C_LIO_LILysis assay performed to quantify host lysis to evaluate viral infection efficiency. C_LIO_LICryo-focused ion beam milling enabled precise preparation of cellular cross-sections for structural analysis. This allows identification of key stages of the {Phi}8 life cycle during the infection cycle. C_LIO_LIThe majority of complete virions were localized extracellularly, emphasizing the role of extracellular stages in the {Phi}8 lifecycle. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/678632v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@6ba760org.highwire.dtl.DTLVardef@1b99d2dorg.highwire.dtl.DTLVardef@fdfe72org.highwire.dtl.DTLVardef@14c077e_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Kumar, S., Robinson, C. F., Bobe, D., Khayat, R.. 2025-09-30. Understanding the infection mechanism of {Phi}8 using infection method in Pseudomonas syringae pv. Phaseolicola strain: In-situ CryoET analysis. https://doi.org/10.1101/2025.09.29.678632

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