Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial compounds and associate with the nucleus through tubular extensions called stromules. Although it is well established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1)-mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector-mediated suppression of BAK1-mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant–pathogen interactions.Fil: Savage, Zachary. Imperial College London; Reino UnidoFil: Duggan, Cian. Imperial College London; Reino UnidoFil: Toufexi, Alexia. Imperial College London; Reino UnidoFil: Pandey, Pooja. Imperial College London; Reino UnidoFil: Liang, Yuxi. Imperial College London; Reino UnidoFil: Segretin, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Yuen, Lok Him. Imperial College London; Reino UnidoFil: Gaboriau, David C. A.. Imperial College London; Reino UnidoFil: Leary, Alexandre Y.. Imperial College London; Reino UnidoFil: Tumtas, Yasin. Imperial College London; Reino UnidoFil: Khandare, Virendrasinh. Imperial College London; Reino UnidoFil: Ward, Andrew D.. Science and Technology Facilities Council; Reino UnidoFil: Botchway, Stanley W.. Science and Technology Facilities Council; Reino UnidoFil: Bateman, Benji C.. Science and Technology Facilities Council; Reino UnidoFil: Pan, Indranil. Alan Turing Institute; Reino Unido. Imperial College London; Reino UnidoFil: Schattat, Martin. Martin Luther Universitat Halle-Wittenberg; AlemaniaFil: Sparkes, Imogen. University of Bristol; Reino UnidoFil: Bozkurt, Osman Tolga. Imperial College London; Reino Unid

Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress.

Funder: Medical Research CouncilSince the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events - e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules