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The function of membrane tethering in plant intercellular communication

Periodic Reporting for period 2 - BRIDGING (The function of membrane tethering in plant intercellular communication)

Reporting period: 2019-12-01 to 2021-05-31

Intercellular communication is critical for multicellularity and evolution gave rise to distinct mechanisms to facilitate this process. Plants have evolved remarkable cellular machines -the Plasmodesmata (PD) pores- which interconnect virtually all cells within the plant body, establishing direct membrane and cytoplasmic continuity, a situation unique to plants. PD are decisive for development, environmental adaptation, defence signalling, and spreading of viruses, yet their mode of action remains elusive.
A striking feature of PD organisation, setting them apart from animal cell junctions, is a strand of endoplasmic reticulum (ER) running through the pore, tethered extremely tight (~10nm) to the plasma membrane (PM) by unidentified “spokes”. To date, the function of ER-PM contacts at PD remains a complete enigma. We don’t know how and why the two organelles come together at PD cellular junctions.
The focus of the ERC-BRIDGING is to understand the function and molecular mechanisms governing ER-PM organelle contacts at PD. We are using multidisciplinary approaches from lipidomics/proteomics, super-high-resolution microscopy, biophysics, molecular dynamics and cell biology and plant genetics to address the following objectives:
• Identify the mechanisms of PD membrane-tethering at the molecular level
• Elucidate the dynamics and 3D architecture of ER-PM contact sites at PD
• Uncover the function of ER-PM apposition for plant intercellular communication.
The protein machinery acting at the ER-PM interface within PD has remained unknown despite more than 40 years of intense research. Combining proteomics, live cell imaging, super-resolution microscopy, molecular dynamics, plant genetics and yeast complementation, we identified the Multiple C2 domains and Transmembrane region Protein (MCTPs) family as the main ER-PM tethers of specialised PD membrane contact sites. We showed show that MCTPs 1) are core PD components and enriched in PD with extremely tight membrane tethering, 2) are critical to plant development and cell-to-cell trafficking 3) insert in the ER through a C-terminal transmembrane region and dock to the PM through several N-terminal C2 domains, through anionic lipid interaction. Our work unveils, for the first time, the molecular identity of PD ER-PM tethers (Brault, Petit et al. 2019, EMBO reports).

How are PD built and organised within the narrow cytoplasmic sleeve between the ER and the PM, and the impact on cell-cell trafficking remained little understood. We used electron tomography to provide the first data of PD ER-PM contact site 3D organisation. Our data showed that within the PD pores, ER-PM contacts undergoes substantial modification and mature from very tight contacts to intermembrane gaps of about 10 nm and spanned by tethers, setting apart two PD morphotypes (Type I and Type II). In collaboration with the team of Yrjö Helariutta’s, we showed that this transition is related to lipid metabolism (Yan et al. 2019, Nature Plants). Phloem Unloading Modulator (plm) is involved in sphingolipid metabolism and plm loss-of-function Arabidopsis mutants present higher phloem unloading capacity in the roots. This increase in PD connectivity was linked with a defect in the remodelling of ER-PM junction from Type I to Type II. This work presents a new paradigm by showing that PD with tight ER-PM contacts may be more conductive than ‘open-sleeved’ ones, challenging current models of how plant cell-cell communication is regulated.

We demonstrated that, similar to other eukaryotic membrane contact sites, PD can rapidly change their molecular composition in response to abiotic stresses to induce cellular responses. From a PD proteomic screen, we identified two LRR-receptor-like kinases, Qian Shou Kinase 1 and Inflorescence Meristem Kinase, which upon osmotic and ionic stresses rapidly re-organised from the PM to PD ER-PM junctions. This process happens remarkably fast, within less than two minutes and is influenced by QSK1 phosphorylation status (Grison et al. 2019 Plant Physiology)
Within the frame of the ERC-BRIDGING project we expect to reveal how ER-PM membrane contact sites are established and controlled at plasmodesmata, and how they contribute to cell-cell communication in plants.