Biology subjects

BOUTTE, Y.

Publications and source records attributed to BOUTTE, Y..

3 recordsLinked to original sources

bioRxiv2025

Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

HIR2 is a plant-specific protein belonging to the superfamily of SPFH domain-containing proteins that were proposed to play scaffolding functions in membranes. HIR2 organizes in plasma membrane (PM) nanodomains that correspond to nanometric scale structures enriched in specific lipids and proteins acting as signaling/regulation hubs. So far, how PM nanodomains are formed and maintained in plant cells remains largely unknown. Combining state of the art microscopy techniques, we investigated the mechanisms governing the trafficking and the organization into nanodomains of Arabidopsis HIR2 protein. We revealed that the mono S-acylation of HIR2 either on C6 or on C7 was required for HIR2 targeting to the PM of Arabidopsis cells, independently of the conventional secretory pathway. Investigating the mechanisms implicated in the arrangement of HIR2 into nanodomains, we provided evidences that the lipid composition in sterols and very long chain fatty acids of the PM influenced HIR2 organization. HIR2 forms oligomers and we demonstrated here that the C-terminal part of HIR2 is required for self-assembly, similarly to animal SPFH proteins. Interestingly, we highlighted that the oligomerization of HIR2 is essential for its organization in nanodomains and to ensure HIR2 lateral stability in the PM. Overall, we propose that HIR2 nanodomain organization is a complex mechanism relying on different parameters including PM lipid composition and oligomerization. HIR proteins are involved in plant immunity. Here, we revealed that HIR2 nanodomain organization is required to boost the apoplastic ROS burst induced by the bacterial peptide flg22. One sentence summaryS-acylation and oligomerization control the plasma membrane targeting and the organization into nanodomains of the Arabidopsis SPFH-domain containing protein HIR2, respectively.

plant biology↗

bioRxiv2025

Identification of INOSITOL PHOSPHORYLCERAMIDE SYNTHASE 2 (IPCS2) as a new rate-limiting component in Arabidopsis pathogen entry control

SIGNIFICANCE STATEMENTPolarized transport of executive defense gene products to sites of attempted microbial invasion is important for plant pathogen entry control and disease resistance. Here, we provide evidence that INOSITOL PHOSPHORYLCERAMIDE SYNTHASE 2 (IPCS2)-dependent sphingolipid production contributes to the role of the trans-Golgi network as a multi-domain sorting compartment, and mediates proper delivery of an ATP-binding cassette transporter to polarized plasma membrane domains at plant-microbe interaction sites. INOSITOL PHOSPHORYLCERAMIDE SYNTHASE 2 (IPCS2) is involved in the biosynthesis of complex sphingolipids at the trans-Golgi network (TGN). Here, we demonstrate a role of IPCS2 in penetration resistance against non-adapted powdery mildew fungi. A novel ipcs2W205* mutant was recovered from a forward genetic screen for Arabidopsis plants with enhanced epidermal cell entry success of the non-adapted barley fungus Blumeria graminis f. sp. hordei (Bgh). A yeast complementation assay and a sphingolipidomic approach revealed that the ipcs2W205* mutant represents a knock-out and lacks IPCS2-specific enzymatic activity. Further mutant analyses suggested that IPCS2-derived glycosyl inositol phosphorylceramides (GIPCs) are required for cell entry control of non-adapted fungal intruders. Confocal laser scanning microscopy (CLSM) studies indicated that upon pathogen attack, IPCS2 remains at the TGN to produce GIPCs, while focal accumulation of the defense cargo PENETRATION 3 (PEN3) at Bgh penetration sites was reduced in the ipcs2W205* mutant background. Thus, we propose a model in which sorting events at the TGN are facilitated by complex sphingolipids, regulating polar secretion of PEN3 to host-pathogen contact sites to terminate fungal ingress.

plant biology↗

bioRxiv2024

Root Expansion Microscopy (ROOT-ExM): A streamlined super resolution method for plants

Expansion microscopy (ExM) has revolutionized biological imaging by physically enlarging samples, surpassing the light diffraction limit and enabling nanoscale visualization using standard microscopes. While extensively employed across a wide range of biological samples, its application to plant tissues is sparse. In this work, we present ROOT-ExM, an expansion method suited for stiff and intricate multicellular plant tissues, focusing on the primary root of Arabidopsis thaliana. ROOT-ExM achieves isotropic expansion with a fourfold increase in resolution, enabling super-resolution microscopy comparable to STimulated Emission Depletion (STED) microscopy. Labelling is achieved through immunolocalization, compartment-specific dyes, and native fluorescence preservation, while N-Hydroxysuccinimide (NHS) ester-dye conjugates reveal the ultrastructural context of cells alongside specific labelling. We successfully applied ROOT-ExM to image various cellular structures, including the Golgi apparatus, the endoplasmic reticulum, the cytoskeleton, and wall-embedded structures such as plasmodesmata. When combined with lattice light sheet microscopy (LLSM), ROOT-ExM achieves 3D quantitative analysis of nanoscale cellular process, revealing increased vesicular fusion in close proximity of the cell plate during cell division. Achieving super-resolution fluorescence imaging in plant biology remains a formidable challenge. Our findings underscore that ROOT-ExM provides a remarkable, cost-effective solution to this challenge, paving the way for unprecedented insights into plant cellular subcellular architecture. One sentence summaryROOT-ExM achieves super-resolution expansion microscopy in plants

plant biology↗