# Plasmodesmata act as unconventional membrane contact sites regulating intercellular molecular exchange in plants

Jessica Perez-Sancho, Marija Smokvarska, Gwennogan Dubois, Marie Glavier, Sujith Sritharan, Tatiana Moraes, Hortense Moreau, Victor Dietrich, Matthieu Platre, Andrea Paterlini, et al.

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## To cite this version:

Jessica Perez-Sancho, Marija Smokvarska, Gwennogan Dubois, Marie Glavier, Sujith Sritharan, et al. Plasmodesmata act as unconventional membrane contact sites regulating intercellular molecular exchange in plants. Cell, 2025, 188 (4), pp.958-977.e23. 10.1016/j.cell.2024.11.034. hal-04963028

HAL Id: hal-04963028

https://hal.inrae.fr/hal-04963028v1

Submitted on 3 Nov 2025

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Distributed under a Creative Commons Attribution 4.0 International License
Plasmodesmata act as unconventional membrane contact sites regulating
inter-cellular molecular exchange in plants

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Jessica Pérez-Sancho#¹, Marija Smokvarska#¹, Gwennogan Dubois#², Marie Glavier¹, Sujith Sritharan³, Tatiana Souza Moraes¹, Hortense Moreau¹, Victor Dietrich¹, Matthieu Pierre Platre⁴, Andrea Paterlini¹,⁵,⁶, Ziqiang Patrick Li¹, Laetitia Fouillen¹, Magali S. Grison¹, Pepe Cana-Quijada¹, Françoise Immel¹, Valerie Wattelet¹, Mathieu Ducros⁷, Lysiane Brocard⁷, Clément Chambaud¹,⁷, Yongming Luo⁸,⁹, Priya Ramakrishna¹⁰,¹¹, Vincent Bayle², Linnka Lefebvre-Legendre¹⁰, Stéphane Claverol¹², Matej Zabrady¹³,¹⁴, Wolfgang Busch⁴, Marie Barberon¹⁰, Jens Tilsner¹³,¹⁴, Yrjö Helariutta⁵,¹⁵, Eugenia Russinova⁸,⁹, Antoine Taly³, Yvon Jaillais²*, Emmanuelle M. Bayer¹,¹⁶*.

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¹ Laboratoire de Biogenèse Membranaire, UMR5200, CNRS, Université de Bordeaux, Villenave d'Ornon, France.

² Laboratoire Reproduction et Développement des Plantes, ENS de Lyon, CNRS, INRA, F-69342 Lyon, France.

³ Laboratoire de Biochimie Théorique, UPR9080, CNRS, Université Paris Cité, Paris, France.

⁴ Salk Institute For Biological Studies, Plant Molecular and Cellular Biology Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.

⁵ The Sainsbury Laboratory, University of Cambridge, Cambridge, UK.

⁶ Present address: Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, UK.

⁷ Bordeaux Imaging Centre, Plant Imaging Platform, UMS 3420, INRA-CNRS-INSERM-University of Bordeaux, Villenave-d'Ornon, France.

⁸ Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium.

⁹ Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium.

¹⁰ Department of Plant Sciences, University of Geneva, 1211 Geneva, Switzerland

¹¹ Present address: Laboratory for Biological Geochemistry, School of Architecture, Civil and Environmental Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015, Lausanne, Switzerland.

¹² Univ. Bordeaux, Bordeaux Proteome, Bordeaux, France

¹³ Biomedical Sciences Research Complex, University of St Andrews, Fife KY16 9ST, UK

¹⁴ Cell and Molecular Sciences, The James Hutton Institute, Dundee DD2 5DA, UK

¹⁵ Institute of Biotechnology, HiLIFE/Organismal and Evolutionary Biology Research Programme, Faculty of Biological and Environmental Sciences, Viikki Plant Science Centre, University of Helsinki, Helsinki, Finland.

¹⁶ Lead contact: emmanuelle.bayer@u-bordeaux.fr (E.M.B.)

39

40 # These authors equally contributed to the work

41 * Correspondence: yvon.jaillais@ens-lyon.fr (Y.J.), emmanuelle.bayer@u-bordeaux.fr (E.M.B.)

42 In brief
43 Plant intercellular communication is regulated via tubular membrane contact through PI4P
44 binding-ER-PM tether MCTP proteins

45
46 Highlights (4 highlights / 85 characters max)

47 1- Plasmodesmata are unconventional ER/PM tubular contact sites located at cell-cell interface

48 2- Plasmodesmata operate as control valves, modulating ER-PM contacts to regulate transport

49 3- MCTP3, MCTP4, MCTP6 and PI4P tethering elements act as valve regulators

50 4- SAC7 PI4P phosphatase controls plasmodesmata MCS in a cell-type-specific manner

51
52
53 SUMMARY
54

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56
57 Membrane contact sites (MCS) are fundamental for intracellular communication, but their role in intercellular communication remains unexplored. We show that in plants, plasmodesmata communication bridges function as atypical endoplasmic reticulum (ER)-plasma membrane
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59 (PM) tubular MCS, operating at cell-cell interfaces. Similar to other MCS, ER-PM apposition is controlled by a protein-lipid tethering complex, but uniquely, this serves intercellular
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61 communication. Combining high-resolution microscopy, molecular dynamics, pharmacological and genetic approaches, we show that cell-cell trafficking is modulated through the combined
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63 action of Multiple C2 domains and transmembrane domain proteins (MCTP) 3, 4, and 6 ER-PM tethers, and phosphatidylinositol-4-phosphate (PI4P) lipid. Graded PI4P amounts regulate MCTP
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65 docking to the PM, their plasmodesmata localization and cell-cell permeability. SAC7, an ER-localized PI4P-phosphatase, regulates MCTP4 accumulation at plasmodesmata and modulates
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67 cell-cell trafficking capacity in a cell-type specific manner. Our findings expand MCS's functions in information transmission, from intracellular to intercellular cellular activities.
68 INTRODUCTION

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107 Recent years have seen a paradigm shift in our understanding of intracellular communication with the discovery of membrane contact sites (MCS), which mediate close contacts between membranes and allow inter-organellar exchange of ions and other molecules (Scorrano et al., 2019; Prinz et al., 2020; Soboloff et al., 2006; Mesmin et al., 2013; Schauder et al., 2014; Chung et al., 2015; von Filseck et al., 2015; Eden et al., 2016; Wong et al., 2016; Wilhelm et al., 2017; Balla, 2018; Bian et al., 2018; Kumagai and Hanada, 2019; Ruiz-Lopez et al., 2021; Qian et al., 2022; Radulovic et al., 2022; Guillén-Samander and De Camilli, 2023; Naón et al., 2023; Wozny et al., 2023). This direct inter-organellar molecular transfer involves specialized protein machinery and membrane proximity regulation (Scorrano et al., 2019). Interest in MCS has grown dramatically in the last decade, with a shift of focus from describing their molecular composition to understanding their functions. Besides inter-organellar communication, MCS are involved in organelle biogenesis, fission and trafficking, cargo sorting, stress responses, and autophagy; highlighting their functional diversity (Eden et al., 2010; Friedman et al., 2011; Hamasaki et al., 2013; Rowland et al., 2014; Pérez-Sancho et al., 2015a; Dong et al., 2016; Hirabayashi et al., 2017; Lees et al., 2017; Collado et al., 2019; Hernández-Alvarez et al., 2019; Hoffmann et al., 2019; Lee et al., 2019a; Wang et al., 2019; Ruiz-Lopez et al., 2021). It is now clear that every organelle inside a single cell forms functional contacts, and that MCS activities are not only broad but also vital for the cell. While MCS are crucial for intracellular activities, multicellular eukaryotes also require communication between individual cells. Currently, our understanding of MCS is limited to intracellular functions, with no evidence that they enable direct molecular exchange between cells.

With this shift in mind, we explored the possible functions of MCS in inter-cellular (as opposed to inter-organellar) molecular exchange. In eukaryotic cells, the endoplasmic reticulum (ER) serves as a central hub for MCS, forming contacts with various organelles (Salvador-Gallego et al., 2017; Wu et al., 2018a). In most eukaryotic cells the ER is confined to a single cell. However, in land plants and some green algae, the ER network extends from cell-to-cell through communication bridges known as plasmodesmata (Nicolas et al., 2017b) (Figure 1A). Inside plasmodesmata the ER form a condensed membrane tube called the desmotubule, which is closely apposed to the PM, creating an atypical tubular MCS-like structure (Ding et al., 1992; Tilsner et al., 2016; Nicolas et al., 2017a). Millions of plasmodesmata weave through the plant body, allowing direct exchange of nutrients and signaling molecules, through the cytoplasmic sleeve, the space between the ER and the PM (Deinum et al., 2019) (Figure 1A). These structures are pivotal for plant intercellular communication, allowing cells to coordinate activities, exchange resources, and respond to environmental and developmental signals (Nakajima et al., 2001; Guseman et al., 2010; Benitez-Alfonso et al., 2013; Faulkner et al., 2013; Stahl et al., 2013; Wang et al., 2013; Daum et al., 2014; Vaddepalli et al., 2014; Otero et al., 2016; Stahl and Faulkner, 2016; Gaudioso-Pedraza et al., 2018; Tylewicz et al., 2018;
108 Miyashima et al., 2019; Song et al., 2019; Tran et al., 2019; Gao et al., 2020; Mellor et al., 2020;
109 Kitagawa et al., 2022; Mehra et al., 2022; Tee et al., 2022; Wang et al., 2023). Yet the molecular
110 mechanisms regulating cell-cell molecular flow remain poorly understood.

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112 While plasmodesmata meet the general criteria of MCS - close membrane proximity, no
113 membrane fusion, specialized function and defined proteome and lipidome (Li et al., 2021), their
114 role as MCS remains unclear. It is still unknown if plasmodesmata-driven cell-cell
115 communication requires regulation of ER-PM contacts and if dysregulation of these impacts
116 plasmodesmata function. To date, local callose synthesis and degradation is the only well-
117 established mechanism regulating plasmodesmata permeability (Zavaliev et al., 2011; Amsbury
118 et al., 2017; Wu et al., 2018b).

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120 Given their position at cell-to-cell interfaces, we hypothesize that regulating ER-PM contact sites
121 at plasmodesmata modulates cytoplasmic diffusion between neighboring cells, representing the
122 first example of MCS-driven intercellular communication in eukaryotes. In this work, we
123 identified the molecular actors of this process, focusing on the root of the model plant
124 *Arabidopsis thaliana*.

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127 RESULTS
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129 MCTP3, MCTP4 and MCTP6 form a tethering complex at plasmodesmata and
130 redundantly impact plant growth and development

131 Plasmodesmata are dynamic nanoscopic structures with the capacity to dilate and
132 contract, acting as checkpoints controlling cell-to-cell trafficking. Sitting at the cell-cell
133 interface, they have the potential to combine membrane contact regulation with intercellular
134 functions. In this scenario, MCS would act as a valve, controlling molecular flow by adjusting
135 the ER-PM gap and/or changing the cytoplasmic sleeve property through steric hindrance
136 (Figure 1A).

137 To test this hypothesis, we first targeted the molecular machinery controlling ER-PM contact,
138 where protein tethers physically bridge the two membranes. We previously identified Multiple
139 C2 domains and transmembrane domain proteins (MCTPs) as putative plasmodesmata tethers
140 (Brault et al., 2019). Miras et al. (Miras et al., 2022b) later confirmed MCTPs as part of 20 core
141 plasmodesmata proteins by cross-referencing proteomes of Arabidopsis, Populus, and Nicotiana.
142 Johnston et al. (Johnston et al., 2023) also classified MCTPs as one of three conserved
143 plasmodesmata families through phyloproteomics

144 We therefore selected MCTPs as ER-PM tether candidates and evaluated their function in cell-
145 cell trafficking in the model plant *Arabidopsis thaliana*.
Structurally similar to animal Extended-synaptotagmins and plant Synaptotagmins, MCTPs consist of a C-terminal ER-anchor and multiple C2 lipid-binding domains (Figure 1B) (Giordano et al., 2013; Pérez-Sancho et al., 2015a; Brault et al., 2019; Ruiz-Lopez et al., 2021). Nearly all plant cells are interconnected by plasmodesmata, and key components are expected to be widely expressed. Among Arabidopsis' 16 MCTP members, MCTP3, MCTP4, and MCTP6 were consistently found in plasmodesmata fractions (Fernandez-Calvino et al., 2011; Kraner et al., 2017; Brault et al., 2019; Kirk et al., 2022; Miras et al., 2022a; Gombos et al., 2023; Johnston et al., 2023) and widely expressed, with MCTP4 showing the highest levels (Figure S1A and S1B).

In all examined tissues, the proteins displayed a dotty localization at cell-cell interfaces, reminiscent of plasmodesmata (Figure 1B; Figure S1C-E; Movie S1-3). All three members show plasmodesmata association when expressed under their endogenous promoters as confirmed by Airyscan imaging and co-localization with callose, a well-established plasmodesmata marker (Figure S1F; Brault et al. 2019). MCTP3, 4, and 6 physically interact when co-expressed, as shown by co-immunoprecipitation in transient expression assays (Figure S2A-B). This interaction at plasmodesmata was confirmed by FRET-FLIM analysis using functional pMCTP4::TagRFP-MCTP4 co-expressed with pMCTP3::eYFP-MCTP3 in the complemented *mctp3-1/mctp4-1/mctp6-1* triple mutant (Figure S2C).

Single mutants (*mctp3-1*, *mctp3-2*, *mctp4-1*, *mctp4-2* or *mctp6-1*) presented no major phenotypic defects (Figure S2D-E, S3). However, double mutants involving MCTP4 (*mctp3-1/mctp4-1*, *mctp3-2/mctp4-1*, *mctp4-1/mctp6-1*) exhibited severe shoot and root growth defects (Figure S2D-E, S3). The triple *mctp3-1/mctp4-1/mctp6-1* mutant was not viable and died after two weeks when grown on soil (Figure S3). The strong pleiotropic phenotype is consistent with a role in the coordination of cellular activity at the multicellular level.

These data indicate that MCTP3, MCTP4, and MCTP6 are core plasmodesmata constituents making them ideal targets for investigating plasmodesmata MCS function.

**MCTPs act as ER/PM tethers inside plasmodesmata bridges, controlling membrane contact**

In previous work, we showed that overexpressing MCTP4 in a delta-ER-PM tether yeast mutant partially restored cortical ER, indicating a potential function as membrane tether (Brault et al., 2019). To test whether MCTP3, MCTP4 and MCTP6 act as ER-PM linkers inside plasmodesmata, we examined whether their genetic deletion would loosen the ER-PM gap within the bridges.
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Functional redundancy has been shown for E-Syts and SYTs (Saheki et al., 2016; Lee et al., 2020; Ruiz-Lopez et al., 2021) with only high-order mutants showing clear ER-PM detachment (Giordano et al., 2013). Therefore, we focused on *mctp3-1/mctp4-1/mctp6-1* mutant for potential plasmodesmata structural defects. We used primary root, enabling high-resolution electron tomography of plasmodesmata in high-pressure frozen, freeze-substituted samples (Nicolas et al., 2017a; Yan et al., 2019). Electron tomography is the only technology capable of achieving the nanometer-scale resolution necessary to visualize plasmodesmata, which are only 20-40 nm in diameter. We focused on the root epidermis division zone, where plasmodesmata structure is well preserved post-cryo-fixation, and on apical-basal walls where plasmodesmata are highly active in transport (Nicolas et al., 2017a). We adapted our previous protocol (Petit et al.; Nicolas et al., 2017a) to reduce staining and increase substructural resolution. This allowed us to visualize the ER-PM boundary within plasmodesmata and discern membrane detachment with greater precision, while maintaining excellent cell preservation (Figure S4A).

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We acquired over 80 tomograms of plasmodesmata. While in wild-type plants, ~70% of plasmodesmata showed tight ER-PM contacts throughout their length (n=50), as previously described (Nicolas et al., 2017a) (Figure 1C, Movie S4). In contrast, only ~7% exhibited this configuration in the *mctp3-1/mctp4-1/mctp6-1* mutant (n=30), with ~93% showing visible ER-PM membrane detachment (Figure 1C (i) (ii), Figure S4B, Movies S5-6). In about ~10% of cases, membrane separation in *mctp3-1/mctp4-1/mctp6-1* was nearly complete (Figure S4B (iii); Movie S6). Additionally, we measured a greater ER-PM distance at detachment points in the triple *mctp* mutant compared to wild-type (Figure 1C (iii)). Average plasmodesmata diameter was slightly higher *mctp* mutant compared to wild-type (Figure S4C) likely due to increased ER-PM detachments.

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These data indicate that MCTP3/MCTP4/MCTP6 act as ER-PM tethers within plasmodesmata bridges, and their loss-of-function results to looser membrane junctions.

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Loss of MCTP ER-PM tethers increases intercellular molecular flow through plasmodesmata

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We next tested the impact of loss of MCTP tethers on intercellular trafficking using fluorescence recovery after photobleaching (FRAP) with the carboxyfluorescein diacetate (CFDA) cytosolic dye. CFDA, a cell-permeant compound, is cleaved by intracellular esterases, forming a membrane-impermeable fluorescent probe that moves from cell-to-cell through plasmodesmata (Nicolas et al., 2017a). After photobleaching in the root division zone, we noted a significantly faster recovery in both *mctp3-2/mctp4-1* and *mctp3-1/mctp4-1/mctp6-1* mutants compared to wild-type (Figure 1D). No differences were noted between the two mutant lines. To validate that loose ER-PM contacts increase molecular flow, we next employed the genetically
encoded DRONPA-s, a reversibly switchable photoactivatable fluorescent marker, with a larger stoke radius (2.8 nm) than CFDA (0.61), allowing us to assess the behavior of molecules of different sizes.

Cytosolic DRONPA-s was activated in a single cell via two-photon microscopy, and movement into neighboring cells monitored (Gerlitz et al., 2018). This enabled us to precisely evaluate variations in molecular flow in apico-basal walls of the root epidermis division zone, where the tomography study was conducted. We selected the double *mntp3-2/mntp4-1* and the triple *mntp3-1/mntp4-1/mntp6-1* mutants to express DRONPA-s. Despite repetitive attempts, we only obtained sufficient DRONPA-s expression levels in the double *mntp3-2/mntp4-1* mutant. After activating DRONPA-s in one cell (N), transport was monitored as the loss of DRONPA-s signal in activated cell or the gain in the neighboring cells (N+1, N+2 and N-1, N-2) (Figure 1E-F). In *mntp3-2/mntp4-1* mutant DRONPA-s loss in the activated cell and movement in the N±1 neighbors was faster (Figure 1E-F; Movie S7), indicating a more permissive plasmodesmata efflux compared to wild-type. This pattern was consistent across the epidermis, cortex and endodermis (Figure 1F).

Together these data show that modifying the ER-PM contacts by means of MCTP3/MCTP4/MCTP6 tether complex, modifies plasmodesmata transport capacity.
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Figure 1. MCTPs function as ER-PM tethers within plasmodesmata, regulating diffusion capacity.

(A) Schematic of ER-PM contact sites in plants.

(B) (i) Schematic of plant ER-PM contact site tethers MCTPs depicting phospholipid-binding C2 domains and ER-anchored transmembrane regions (TMR). Schematic of MCTP ER-PM tethers showing C2 phospholipid-binding domains and ER-anchored transmembrane regions (TMR) (not to scale).

(ii) 3D view of *mctp3/4 pUBQ10::eYFP MCTP3*, *mctp3/4 pUBQ10::eYFP-MCTP4* and *mctp3/4 pUBQ10::MCTP6-mVenus* acquired with lattice light-sheet microscopy. Insets show a single focal plane. In this panel and throughout all the figures in the paper, *mctp3/4* stands for *mctp3-2/mctp4-1* and *mctp3/4/6* stands for *mctp3-1/mctp4-1/mctp6-1*.

(C) (i) Tomographic slices and segmented 3D reconstructions of plasmodesmata in wild-type (WT) and *mctp3/4/6*. Arrows pointing to ER-PM detachment. (ii) Percentage of plasmodesmata showing ER-PM detachment. (iii) Quantification of ER-PM distance at the widest gap in WT and *mctp3/4/6* plasmodesmata.

(D) CFDA mobility assay measured by FRAP in root epidermal meristematic cells in WT, *mctp3/4* and *mctp3/4/6*.

(i)

Representative images before and after the bleach. Cells used for quantification are surrounded by dotted squares.

(ii)

Normalized fluorescence intensity over a period of 90 seconds after bleaching. Half-life (τ) of CFDA recovery for each genotype is shown. (iii) Normalized fluorescence intensity 2 seconds after photobleaching. Statistical analysis was done with ANOVA followed by Tukey's test. Experiment was repeated twice with similar results.

(E-F) DRONPA-s mobility assay measured diffusion from a single activated cell (using two-photon) to the neighboring cells. (E) (i) Representative DRONPA-s images in WT and *mctp3/4* before activation, at activation and 60 and 120 second post-activation. N, activated cell; N±1, direct neighbors; N±2, second level neighbors. Propidium iodide (PI, white) marks cell peripheries. (ii) Signal plot showing raw intensity values from the images in (i). F (i) Normalized DRONPA-s fluorescence over time averaged from N±1 in meristematic epidermis, cortex and endodermis cells. Lines indicate mean. Error bars indicate SEM. (ii) Normalized DRONPA-s fluorescence in the N±1 cells at 120 seconds post activation from (i). At least 6 roots per cell type per genotype, repeated 3 times, data pooled for quantification. When not differently stated, statistical analysis was done with Student t-text. Lines indicate median. Error bars indicate 95% confidence interval (CI).

Heightened cell-to-cell diffusion in *mctp* mutants is independent of callose deposition

In text-book models, cell-cell trafficking is primarily regulated by callose, a beta 1-3 extracellular glucan enriched around plasmodesmata and metabolized by plasmodesmata-located enzymes. Callose accumulation reduces transport while its degradation enhances it (Amsbury et al., 2017). While the exact mechanism of callose action remains elusive, it is proposed to modulate the cytoplasmic sleeve size, by increasing or relaxing pressure on the PM.

We therefore enquired if enhanced molecular flow in *mctp3-2/mctp4-1* and *mctp3-1/mctp4-1/mctp6-1* mutants were due to decreased callose levels. First, we performed RNAseq analysis of root extracts to check if the expression of genes associated with callose regulation at plasmodesmata were modified. The data show that none of the callose-related proteins were misregulated in either single or double *mctp* mutant, compared to wild-type (Figure 2A; Figure S5). We then performed comparative proteomic analysis to assess the potential mis-regulation of callose-related proteins at the protein level. Total protein extract from wild-type, double and triple *mctp* mutant seedlings were analyzed by liquid chromatography-tandem mass spectrometry. The relative enrichment or depletion of proteins in *mctp* mutants versus wild-type was determined (see M&M for details). Using the same protein set from the callose-related
289 RNAseq analysis (Figure 2A), we found no significant changes in detected callose-related
290 proteins (Figure 2B). We next quantified callose levels in situ in the root division zone (same as
291 for DRONPA-s cell-cell trafficking assays) using callose-specific monoclonal antibodies, finding
292 that double and triple *mctp* mutants accumulated callose similarly to wild-type plants (Figure
293 2D). From these data, we concluded that increase of cell-cell trafficking in *mctp* mutants is not
294 due to reduced callose levels.

<figure><img src="image_27.png" /><figcaption>**Figure 2. Loss of function of MCTP does not cause defects in callose deposition**</figcaption></figure>

(A) Normalized expression of callose-related enzymes based on mRNA-seq of WT and *mctp3/4*. FPKM, Fragments per kilobase mapped. Bars reach the value of the median. Error bars represent 95% CI. Statistical analysis was done

295
296 **Figure 2. Loss of function of MCTP does not cause defects in callose deposition**
297 (A) Normalized expression of callose-related enzymes based on mRNA-seq of WT and mctp3/4. FPKM, Fragments
298 per kilobase mapped. Bars reach the value of the median. Error bars represent 95% CI. Statistical analysis was done
299 with multiple paired t-test. Four biological replicates, each being a pool of 40 mg of 5-days-old roots, cut 1 cm from
300 the tip.

301 (B) Normalized protein abundance of callose-related enzymes from mass spectrometry of WT, *mctp3/4* and
302 *mctp3/4/6*. Bars reach the value of the median. Error bars represent 95 % CI. Statistical analysis was done with
303 background-based ANOVA. Three biological replicates, each being a pool of 80 mg of 5-days-old roots, cut 1 cm
304 from the tip.

305 (C) Representative images of whole-mount anti-callose immunostaining (magenta) of WT root apical meristem.
306 Calcofluor (blue) was used to stain cell-walls. The white rectangle indicates the area generally used for
307 quantification. For each mature cell-wall, a segmented line was drawn (dashed-red line as an example) and the mean
308 intensity value along the line was used for quantification. Red arrowheads mark cell plates (showing intense anti-
309 callose signal and weak calcofluor signal) that were excluded from the analysis.

310 (D) Callose accumulation in WT, *mctp3/4* and *mctp3/4/6* epidermis, cortex and endodermis meristematic cells. (i)
311 Representative confocal images of anti-callose immunostaining. (ii) Quantification of callose as explained in (C).
312 Lines indicate median. Error bars indicate 95 % CI. Statistical analysis was done with ANOVA followed by Tukey's
313 test. Data pooled from two experiments with at least four roots per genotype.

314
315
# Defective ER-PM tethering impairs the dynamic control of intercellular molecular flow

316
317 Intercellular communication is dynamically controlled, and a key feature of
318 plasmodesmata lies in their ability to respond to environmental or developmental stimuli by
319 modulating intercellular molecular flow (Gisel et al., 1999; Faulkner et al., 2013; Han et al.,
320 2014; Cui and Lee, 2016; Cheval and Faulkner, 2018; Gaudioso-Pedraza et al., 2018; Lexy et al.,
321 2018; Tylewicz et al., 2018; Grison et al., 2019; Sager et al., 2020; Mehra et al., 2022; Tee et al.,
322 2022; Wang et al., 2023). We therefore tested whether dynamic regulation of trafficking
323 persisted in the absence of a fully functional tether machinery.

323 We first tested plasmodesmata's response to brassinosteroid, which are known to modify cell-
324 cell transport and callose deposition (Wang et al., 2023). We used brassinazole, a brassinosteroid
325 biosynthesis inhibitor, which drastically reduces callose levels and promotes cell-cell trafficking
326 (Wang et al., 2023). After 24 hours of 1 µM brassinazole exposure, wild-type roots showed
327 almost complete suppression of callose accumulation (Figure 3A). A similar effect was observed
328 in *mctp3-2/mctp4-1* and *mctp3-1/mctp4-1/mctp6-1* mutants, reaffirming that callose response
329 remains unaffected in the *mctp* mutants (Figure 3A). As expected, in wild-type plants,
330 DRONPA-s spread faster into neighboring cells after callose removal (Figure 3B), with
331 trafficking levels matching those of untreated *mctp3-2/mctp4-1* plants. However, callose removal
332 in the *mctp3-2/mctp4-1* mutant did not alter macromolecular flux, which remained identical to
333 control conditions (Figure 3B).

334
335 We next tested whether down-regulation of cell-cell trafficking was functional without MCTP
336 tethers, by examining *mctp* mutant responses under conditions that plasmodesmata closure
337 through callose deposition. First, we exposed wild-type plants to brassinolide (24 h, 200 nM) and
338 confirmed plasmodesmata closure (Wang et al., 2023), indicated by reduced DRONPA-s cell-to-
339 cell diffusion and increased callose accumulation (Figure S6A-B). In this condition, *mctp*
340 mutants showed an increase in callose identical to wild-type (Figure S6B). However, the impact
341 on cell-cell trafficking was minor and DRONPA-s diffusion remained at the same permissive
342 level in brassinolide-treated *mctp4-1/mctp3-2* mutant as in mock-treated wild-type plants (Figure
343 S6A). No obvious differences in MCTP4 localisation pattern were observed upon brassinosteroid
344 treatments (Figure S6C).

345 We next used salt stress (15 min, 20 mM), which triggers short-term callose deposition and
346 plasmodesmata closure (Hunter et al., 2019), as confirmed by quantitative callose
347 immunolocalization and DRONPA-s diffusion (Figure 3C-D). Callose deposition were similar
348 in *mctp3-2/mctp4-1*, *mctp3-2/mctp4-1/mctp6-1* and wild-type roots (Figure 3C). However, while
349 wild-type showed sharp decrease in DRONPA-s cell-cell trafficking, plasmodesmata closure was
350 inhibited in *mctp3-2/mctp4-1* mutants (Figure 3D).

351
352
353 Collectively these data indicate that without the MCTP-tether machinery, callose cannot
354 efficiently regulate plasmodesmata. To confirm this, we genetically manipulated callose levels
355 by expressing the gain-of-function callose synthase 3 gene (*cals3m*) (Vatén et al., 2011) under an
356 inducible expression system in epidermal cells in *mctp3-2/mctp4-1* mutant expressing
357 DRONPA-s.

358
359
360
361
362
363
364
365 After 3h of β-estradiol induction, we observed significant callose accumulation in both the wild-
type and *mctp3-2/mctp4-1* mutants (Figure 3E). Cals3m-induced callose deposition translated
into a drastic reduction in DRONPA-s cell-cell trafficking in the wild-type but not in the *mctp3-
2/mctp4-1* mutant (Figure 3F, Movie S8).

Altogether, these data indicate that 1) dynamic regulation of cell-cell molecular flow is lost in the
absence of functional ER-PM tethers, and 2) callose regulation is no longer effective in *mctp*
mutants.
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**Figure 3.** Callose regulation in loss of function MCTPs has minor effects on the cell-cell diffusion

(A-B) Brassinazole treatment; DMSO or 1 µM Brassinazole (BRZ) for 24 h. (A) Anti-callose immunostaining in treated WT, *mctp3/4* and *mctp3/4/6*. (i). Representative confocal images of callose immunostaining. (ii) Quantification of callose. (B) DRONPA-s mobility assay in treated WT and *mctp3/4*. (i) Representative images of DRONPA-s diffusion. (ii) Normalized DRONPA-s fluorescence over time. Line show mean. Error bars show SEM. (iii). Percentage of DRONPA-s normalized fluorescence in the N±1 cells at 120 s post activation from (ii).

(C-D) Short-term salt treatment; H2O (control) or 20mM NaCl for 15 min. (C) Anti-callose immunostaining in treated WT, *mctp3/4* and *mctp3/4/6* (i) Representative confocal images of callose immunostaining. (ii) Quantification of callose. (D) DRONPA-s mobility assay in treated WT and *mctp3/4*. (i) Representative images of DRONPA-s diffusion. (ii) Normalized DRONPA-s fluorescence over time. Line show mean. Error bars show SEM. (iii) Mean percentage of DRONPA-s normalized fluorescence in the N±1 cells at 120 s post activation from (ii).

(E-F) Callose-inducible *pPRD9::XVE-cals3m*; DMSO or 10µM b-estradiol for 3 h. (E) Anti-callose immunostaining. (i) Representative confocal images of callose immunostaining. (ii). Quantification of callose. (F) DRONPA-s mobility assay. (i) Representative images of DRONPA-s diffusion. (ii) Normalized DRONPA-s fluorescence over time. Line show mean. Error bars show SEM. (iii) Percentage of DRONPA-s normalized fluorescence in the N±1 cells at 120 s post activation from (ii).

When not specified differently, lines indicate median. Error bars indicate 95 % CI. Statistical analysis was done with ANOVA followed by Tukey's test. Different letters indicate p<0.05.

A minimum of 5 roots were used per genotype, per condition, per experiment. Experiments were repeated three times with similar results and data were pooled.

**PI4P levels control MCTP docking to the PM**

MCTPs are anchored into the ER through their C-terminal transmembrane region, and were proposed to dock to the PM by interacting with anionic phospholipids through their C2-lipid binding domains (Brault et al., 2019) similar to E-Syts and SYTs (Giordano et al., 2013; Pérez-Sancho et al., 2015a; Benavente et al., 2021) (Movie S9). Anionic phospholipids act as signals in membranes, being tightly regulated during development, reproduction and environmental interactions. We investigated whether anionic lipids could regulate plasmodesmata by controlling MCTP interaction with the PM, focusing on PI4P, a key determinant of the PM's inner leaflet electrostatic signature in plants (Simon et al., 2016a).

Previous studies indicated that PI4P may promote docking of C2's to the PM (Brault et al., 2019). To confirm these data, we purified individual C2 domains of MCTP4. While C2C and C2D were insoluble after expression in bacteria, we successfully purify C2B and confirmed its preferential binding to liposomes containing PI4P (Figure 4A). While PI4P is present in the bulk PM, plasmodesmata, like other MCS, display a specialized membrane environment (Li et al., 2021) and it is not known whether they contain phosphoinositide species. We thus purified plasmodesmata from *A. thaliana* cell culture and conducted LC-MS/MS analysis, which detected both PIP and PIP2 species in plasmodesmata fractions (Figure 4B).

We next explored PI4P's role in MCTP membrane tethering using molecular modeling and dynamics for near-atomistic insights into MCTP4's interaction with the PM, using Martini 3
coarse-grained force field (Souza et al., 2021; Borges-Araújo et al., 2023) and Alphafold2
structure prediction (Jumper et al., 2021).

Previous work, focused on individual C2 domains (Brault et al., 2019), here we analyze the full
MCTP4 cytosolic domain (excluding the ER-anchored transmembrane region) in the presence of
a PM-like lipid bilayer. We restricted MCTP4's movement to simulate realistic protein
positioning relative to the PM, so that the C-terminus of the polypeptide (where the TMR
normally is) could not face the PM (Figure 4C). The structural model of MCTP4's cytosolic
part, with three C2 domains, was extracted from (Sritharan et al., 2023) and embedded in a water
box with a lipid bilayer mimicking the PM (~70% phosphatidylcholine (PC), 20% Sitosterol,
10% phosphatidylserine (PS), 2% PI4P). To assess MCTP4's interaction with the PM, we tracked
fluctuating distances between the C2 domains and the lipid bilayer over time (Figure 4D) and
mapped the distribution of distances as a function of sequence (y axis) and time (x axis) (Figure
4E). Interactions with the PM involve various docking configurations with one C2 (C2C Figure
4F state3 or C2D Figure 4F state4), two C2s (C2B and C2C) (Figure 4F state1), or all three
(Figure 4F state2) interacting (Movie S10). We next evaluated MCTP4's interaction with the
PM with varying PI4P concentrations (0%, 2% or 6%). The data indicate that PI4P increases C2s
binding frequency, suggesting a stabilizing role in PM docking (Figure 4G).

We then identified the amino acids interacting with PI4P. Examining the electrostatic profile of
the C2 domains revealed several positively charged regions mainly associated with lysines
(shown in Movie S11 for green-bordered lysine residues) which are important for membrane
binding (Tubiana et al., 2022).

Simulations with 2% PI4P showed significant interactions with the lipid bilayer of these regions
(Figure S7A). Focusing on the C2C domain which presented the highest hit targets and we
identified five residues (D217, K218, R220, K318, K322) with high PI4P-interaction frequency
(Figure 4I and S7B). After mutating them to reduce their electrostatic potential (Figure 4H and
S7B), simulations showed reduced membrane contacts for the C2C domain, even in the presence
of PI4P (Figure 4J)

Altogether, our data show that MCTP C2 domains interact with membranes conditionally based
on PI4P concentration, a lipid present at plasmodesmata. This suggests that PI4P, as a signaling
lipid, may regulate MCTP's interaction with the PM.
<figure><img src="image_40.png" /><figcaption>Figure 4. PI(4)P regulates MCTP4 docking to the PM via C2-lipid binding domains</figcaption></figure>

(A) Liposome binding assay of the purified MCTP4 C2B domain. (i). Schematic representation of the assay, with colored liposomes representing different composition. (ii) SDS-PAGE showing C2B in supernatant (sup.) and precipitated (pellet) fractions after centrifugation. Proteins were visualized with a Bio-Rad stain-free imager. One single band was observed per well, corresponding to MCTP4 C2B domain. (iii) Quantification of MCTP4 C2B

445

446

447

448

449

450
binding to liposomes of different lipid compositions. Bound fraction was calculated as the ratio of C2B in the pellet to C2B in the supernatant. Line indicates median. Error bars indicate 95% CI. Statistical analysis was done using ANOVA followed by Tukey test. Different letters indicate p<0.05. Experiment was repeated 3-4 times and data were pooled together. PC: phosphatidyl choline; PS: phophatidyl serine; PI4P: phosphatidyl inositol 4-phosphate; PA: phosphatidic acid; PI(4,5)P2: phosphatidyl inositol 4,5-biphosphate.

(B) Identification of phosphoinositides in plasmodesmata purified from WT cell cultures (4 biological replicates).

(C) Application of the Colvar Tilt variable to MCTP4. The harmonic potential activates when the tilt angle is below 0, ensuring the system maintains a realistic orientation relative to the membrane even without TMR/ER

(D) Analysis of the minimum distance between C2 domains and the lipid bilayer over 3 microseconds, illustrating the dynamics of domain-membrane interactions from a single simulation.

(E) Heatmap of C2 domain-PI4P distance from simulations in (C). The distances are color-coded according to the legend in the right.

(F) Snapshots from a simulation of the MCTP4 cytoplasmic domain interacting with a PM-like lipid bilayer containing 2% PI4P: (1) Interaction via two C2 domains, (2) All three C2 domains interacting with the bilayer simultaneously, and (3 & 4) interaction involving one C2 domain. The simulations lasted 3 microseconds and were repeated 5 times for each condition. The C2 domains are colored as follows: C2B in magenta, C2C in green, and C2D in cyan.

(G) Histograms comparing the interaction frequency (average global contact) under three PI4P conditions: without PI4P, with 2% PI4P, and with 6% PI4P, showing increased frequency and stabilization of MCTP4-PM interactions with higher PI4P concentrations.

(H) Surface representation of the C2 block with electrostatic potential ranging from -5 (red) to 5 (blue), created using ChimeraX. The two angled views illustrate the differences between the WT and the C2 block with mutated amino acids on C2C, which are putatively responsible for binding with PI4P.

(I) Schematic of the C2 block (C2B in magenta, C2D in cyan) focusing on C2C (in green) highlighting mutated amino acids (in dark blue).

(J) Average PM contact counts of the three separate C2 blocks under conditions with no added PI4P, 2% PI4P and when putative lipid binding amino acids are mutated on the C2C block in 2% PI4P. Error bars show SD. Data from 7 simulations.

(J) Average PM contact counts of the three separate C2 blocks under conditions with no added PI4P, 2% PI4P, and with mutations in putative lipid-binding amino acids on the C2C block in 2% PI4P. Error bars represent SD. Data from 7 simulations.

MCS modulates cell-cell molecular flow through the tandem action of MCTP tethers and PI4P lipid.

We next tested the impact of PI4P on cell-cell trafficking. Our data supports a scenario where MCTPs bind to the PM through interaction with PI4P. Thus, the removal of PI4P should phenocopy *mctp3-2/mctp4-1* in terms of cell-cell transport capacity. To test this hypothesis, we assessed the impact of reduced PI4P levels by applying the PI4K inhibitor PAO (phenylarsine oxide) at a concentration and exposure time known to affect PI4P sensors localization in Arabidopsis roots (Simon et al., 2016) (Figure 5A).

In contrast, YM201636 (PI(3,5)P₂ synthesis inhibitor) and Wortmannin (PI3P synthesis inhibitor) did not change plasmodesmata trafficking (Figure S8A-C). We then subjected *mctp3-2/mctp4-1 p35S::DRONPA-s* to the same PAO treatment, finding no effect on the mutant's
transport capacity (Figure 5A), supporting that MCTP and PI4P act together to regulate plasmodesmata. Neither PAO nor DMSO differentially affected callose levels in wild-type and *mctp3-2/mctp4-1* mutant backgrounds (Figure 5B).

If PI4P levels regulate cell-cell trafficking, elevated PI4P should induce plasmodesmata closure. Therefore, we next assayed if increased PI4P level modifies cell-cell transport. To do so, we generated a stable line expressing DRONPA-s in the *suppressor of actin7* (*sac7*, or *root hair defective4*) mutant. SAC7 is a PI4P 4-phosphatase, whose mutant accumulates PI4P *in vivo* (Thole et al., 2008; Song et al., 2021). The *sac7* showed reduced DRONPA-s diffusion, supporting the role of PI4P in regulating plasmodesmata connectivity (Figure 5C). Compared to the wild-type, *sac7* has decreased accumulation of callose, ruling out callose as the cause of plasmodesmata closure (Figure 5D).

We then asked whether callose removal could still open plasmodesmata with increased PI4P levels. Therefore, we treated p35S::DRONPA-s lines with brassinazole (24h, 1 µM), which led to callose removal in both wild-type and *sac7* backgrounds (Figure 5F). However, while wild-type roots responded by opening plasmodesmata with faster DRONPA-s movement, *sac7* maintained plasmodesmata closed (Figure 5E). Thus, the signaling lipid PI4P modifies cell-to-cell cytoplasmic diffusion bypassing callose accumulation, with reduced PI4P levels enhancing molecular movement, and elevated levels diminishing flow. From these data we concluded that the signaling lipid PI4P act as a regulator of plasmodesmata connectivity.
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<figure><img src="image_50.png" /><figcaption>**Figure 5:** PI4P levels modulate cell-cell molecular flow.

(A-B) PAO treatment (DMSO or PAO 60 nM, 30 min). (A) DRONPA-s diffusion assay in PAO-treated WT and *mctp3/4*. (i) Phosphoinositide metabolism schema showing PAO blocking PI4K. (ii) Representative DRONPA-s diffusion images. (iii) Normalized DRONPA-s fluorescence over time. Line indicates mean. Bars show SEM. (iv) Percentage of DRONPA-s normalized fluorescence in the N±1 cells at 120 seconds post-activation from (iii). (B) Anti-callose immunostaining quantification in WT, *mctp3/4* and *mctp3/4/6*.

(C-D) *sac7-1* mutant. (C) DRONPA-s diffusion assay in WT and *sac7-1*. (i) Phosphoinositide metabolism schema showing SAC7 as PI4P phosphatase. (ii) Representative DRONPA-s diffusion images. (iii) Normalized DRONPA-s fluorescence over time. Line indicates mean. Bars show SEM. (iv) Percentage of DRONPA-s normalized fluorescence in the N±1 cells at 120 seconds post-activation from (iii). Statistical analysis was done using Student t-test. (D) Anti-callose immunostaining quantification in WT and *sac7-1*. Statistical analysis was done using Student t-test</figcaption></figure>
532 (E-F) *sac7-1* mutant, BRZ treatment; DMSO or 1 µM BRZ for 24 h (A) DRONPA-s mobility assay in treated WT
533 and *sac7-1*. (i) Representative images of DRONPA-s diffusion. (ii) Normalized DRONPA-s fluorescence over
534 time. Line show mean. Error bars show SEM. (iii) Percentage of DRONPA-s normalized fluorescence in the N±1
535 cells at 120 s post activation from (ii). (F) Quantification of anti-callose immunostaining in treated WT and *sac7-1*.
536 When not differently stated, statistical analysis was done using ANOVA followed by Tukey's test. Lines indicate
537 median. Error bars indicate 95% CI. Different letters indicate p<0.05. A minimum of 5 plants per genotype, per
538 condition, per experiment were used. Experiment were repeated 3 times and data were pooled together.
539

540 SAC7 PI4P phosphatase is a cortical ER protein that closely associates with plasmodesmata
541 in a cell type specific manner

542
543 Our data suggest that graded PI4P levels modulate plasmodesmata connectivity. We then
544 examined the mechanistic basis of this regulation and whether variations of PI4P levels are
545 relevant to regulate plasmodesmata connectivity in a wild-type situation. To this end, we focused
546 on the SAC7 enzyme, because i) the *sac7* mutant showed impaired plasmodesmata connectivity
547 and ii) SAC7 was identified in our plasmodesmata proteome (Brault et al., 2019).
548

549 To analyze SAC7 expression pattern and localization, we transformed the *sac7* mutant with
550 pSAC7::mCitrine-SAC7 and pSAC7::2xmCherry-SAC7 constructs. Both fusion proteins
551 complemented *sac7* mutant's short root hair phenotype, indicating functionallity (Figure S9 A-
552 B).

553 Looking at SAC7 localization in root, we noticed a distinct tissue-specific expression pattern,
554 with SAC7 predominantly expressed in the root hair cell file (trichoblast) and at much lower
555 levels in non-root hair cells (atrichoblast) (Figure 6A-B). To confirm SAC7's strong expression
556 in trichoblast cells, we crossed the pSAC7::2xmCherry-SAC7 line with the EXP7 SAND reporter
557 line, which is exclusively expressed in trichoblasts (Marquès-Bueno et al., 2016). Colocalization
558 revealed that 2xmCherry-SAC7 was indeed preferentially expressed in trichoblast, and present in
559 the meristem before the bulging and elongation of root hairs (Figure 6B).

560
561 We next analyzed the subcellular localization of pSAC7::2xmCherry-SAC7. The protein
562 accumulated at the tip of growing root hairs, as previously reported (Figure S9C) (Thole et al.,
563 2008). SAC7 also localized in reticulated structures resembling ER membranes. Given SAC7 ER
564 localization in BY-2 cells and that its orthologs, Sac1p/SAC1, are ER-resident proteins (Dubois
565 and Jaillais, 2021), we examined whether SAC7 could locate in this compartment in Arabidopsis.
566 In cotyledon epidermis cells, characterized by a reticulated ER, mCitrine-SAC7 colocalized with
567 the ER marker DDRGK1 Domain-containing protein 1-mCherry (Figure S9D) (Stephani et al.,
568 2020). In the root meristem, SAC7 also displayed an ER-localisation pattern while exhibiting a
569 pronounced enrichment at the apico-basal cortical region of the cell (Figure 6C).

570
571 To gain insights into this unique localization pattern, we compared SAC7 (mCitrine-SAC7 and
572 2xmCherry-SAC7) with various ER markers, including the cortical ER marker SYT1-GFP, the
573 bulk ER membrane proteins DDRGK1-mCherry and Cinnamate-4-hydroxylase-GFP (C4H-GFP)
and the ER luminal marker RFP-HDEL (Figure 6D). We quantified the cortical enrichment of SAC7, using Apico-basal vs Intracellular Fluorescence ratio (APIFR) for all ER proteins (Figure 6E). SYT1, an ER-PM tether (Pérez-Sancho et al., 2015b), showed similar enrichment in the cortical ER as SAC7 (Figure 6D-E) but unlike SAC7, also accumulated at the lateral sides of atrichoblast cells (Figure 6D). The other ER proteins displayed a more uniform distribution without significant cortical enrichment and a prominent labeling of the perinuclear ER (Figure 6D-E). These results highlight a unique polar localization of SAC7 in the apical-basal cortical ER membranes.

Previous reports indicated that plasmodesmata are enriched at the apico-basal cell-cell interface rather than lateral sides, in Arabidopsis root epidermis (Zhu et al., 1998). We therefore checked SAC7 association with MCTP4. Using high-resolution airyscan microscopy, we colocalized 2xmCherry-SAC7 with GFP-MCTP4 both expressed under their native promoters. We found that 2xmCherry-SAC7 accumulated near GFP-MCTP4-labeled plasmodesmata (Figure 6F). Thus, SAC7 is an ER protein, enriched in the apico-basal cortical ER of trichoblast cells, in close association with plasmodesmata. These data suggest that the SAC7 PI4P phosphatase may regulate PI4P levels at or near plasmodesmata.
<figure><img src="image_51.png" /><figcaption>Figure 6: SAC7 is preferentially expressed in trichoblast cells and is polarly localized in the ER nearMCTPs</figcaption></figure>

(A) Schematic of the root epidermis showing alternating trichoblasts (T) and atrichoblast (A) cells.

(B) Confocal images showing *pSAC7::2xmCherry-SAC7* expression overlapping with *pEXP7::4xYFP* (trichoblast marker) in root-tip epidermis.

(C) Confocal images of *pSAC7::mCitrine-SAC7* (yellow) co-localizing with the bulk ER marker *pUBQ10::DDRGK1-mCherry* (magenta).

(D) Confocal images of ER-localized proteins in root meristematic cells. White arrows point toward apico-basal accumulation of SAC7 protein.

(E) (i) Apical-basal vs intracellular fluorescent ratio (APIFR). (ii) Quantification APIFR of the proteins shown in (D). Statistical analysis was done with ANOVA followed by Tukey's test. Lines indicate median. Error bars indicate 95% CI.

(F) Confocal airyscan micrographs of roots coexpressing *pSAC7::2xmCherry-SAC7* (magenta) and *pMCTP4::MCTP4-GFP* (yellow) in epidermal cells, with a zoom on the apical-basal cell-cell interface.

SAC7 is a cell-type specific regulator of MCTP localization and plasmodesmata connectivity
Our data indicate that PI4P levels regulate MCTP4 interaction with the PM and cell-cell connectivity. SAC7 PI4P phosphatase localizes near MCTP4, and show high expression in trichoblasts (low PI4P) and low expression atrichoblasts (high PI4P). We thus hypothesized that SAC7 negatively regulate MCTP and cell-to-cell connectivity.

We first analyzed whether GFP-MCTP4 localization when expressed under its native promoter differed between trichoblast and atrichoblast cells in wild-type plants. To this end, we counterstained cell walls using propidium iodine (PI) and automatically segmented the cell's contour. We then quantified the GFP-MCTP4 signal at cell-cell junctions, and found higher MCTP4 accumulation at the apical-basal interface in atrichoblasts (low SAC7) compared to trichoblasts (high SAC7) (Figures 6E, 7A). We next investigated how GFP-MCTP4 differential localization in trichoblasts and atrichoblasts affects cell-to-cell connectivity in wild-type plants by quantifying DRONPA-s diffusion in each cell file. We found that DRONPA-s diffused faster in trichoblast than atrichoblast cells, correlating with high SAC7 expression and low MCTP4 levels in this cell type (Figure 7B).

We then wondered if eliminating SAC7's differential cell type expression would impact MCTP4 localization and thereby cell-cell connectivity. To this end, we expressed both GFP-MCTP4 (under its native promoter) and cytosolic DRONPA-s in *sac7-1* mutant background. We observed a strong increase of GFP-MCTP4 signal at the apico-basal walls of trichoblast cells in *sac7* (Figure 7A). We also found a slight increase of GFP-MCTP4 in *sac7* atrichoblasts compared to wild-type, likely due to the weaker SAC7 expression in this cell type. These changes in MCTP4 localization correlated with reduced cell-cell molecular flow in the *sac7-1* mutant compared to wild-type, particularly affecting the trichoblast cell file (Figure 7B).

We then asked how low PI4P levels would impact plasmodesmata structure using electron tomography on high pressure frozen freeze substituted root samples. Because it is difficult to unambiguously distinguish between trichoblast and atrichoblast cells without cell type markers, we generated SAC7 over-expressing lines (pUBQ10::2XmCherry-Myc-SAC7) and screened the apico-basal walls in the epidermis division zone (as for Figure 1C). Plasmodesmata in these lines presented frequent ER-PM detachment events (~97 % plasmodesmata; n=33) (Figure 7C; Movie S12) similar to *mctp* triple mutants (Figure 1C).

At the root tip, wild-type epidermis trichoblast and atrichoblast cells exhibit varying degrees of cell-to-cell molecular diffusion, with distinct elongation rates, physiological states, and interactions with the underlying cortex. While the physiological implications of this differential connectivity are not yet clear, our data demonstrate that ER/PM crosstalk, through developmental modulation of PI4P/MCTP interactions, can regulate plasmodesmata connectivity.
Our results indicate that graded SAC7 expression impacts MCTP4 localization in a cell-type-dependent manner, directly regulating plasmodesmata connectivity, leading to varying degrees of cell-to-cell molecular diffusion in wild-type epidermis trichoblast and atrichoblast cells at the root tip. Trichoblast and atrichoblast cells exhibit distinct elongation rates, physiological status, interactions with the underlying cortex and overall have specific functions in root biology. While the physiological effect of their differential connectivity remains to be determined, our data serve as a proof-of-concept that an ER/PM crosstalk, via developmental modulation of PI4P/MCTP interactions, can regulate plasmodesmata connectivity.
A (i)

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(ii)

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B (i)

WT p35S:Dronpa-s

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sac7-1 p35S:Dronpa-s

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C (i)

pUBQ10::2XmCherry-Myc-SAC7

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D

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High intercellular molecular flow

* ✓ Loose ER-PM contact

* ✓ Low PI4P

* ✓ Weak MCTPs docking

Low intercellular molecular flow

* ✓ Tight ER-PM contact

* ✓ High PI4P

* ✓ Enhanced MCTPs docking

* ✓ MCTPs accumulation

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**Figure 7: SAC7 regulates MCTP localization and cell-to-cell diffusion in a cell type-specific manner**
(A) APIFR of *pMCTP4::MCTP4-GFP* in WT or *sac7-1* trichoblast (T) and atrichoblast (A) cells. (i) Confocal images showing PI-stained cell peripheries. (ii) APIFR quantification. Statistical analysis was done with ANOVA followed by Tukey's test. Lines indicate median. Error bars indicate 95% CI.
(B) DRONPA-s diffusion assay in WT and *sac7-1* trichoblast and atrichoblast meristematic cells.
(i) Representative images of DRONPA-s diffusion. (ii) Normalized DRONPA-s fluorescence over time. Lines indicate mean. Bars show SEM. (iii) Percentage of DRONPA-s normalized fluorescence in N±1 cells at 120 seconds post activation. Statistical analysis was done with ANOVA followed by Tukey's test. Lines indicate median. Error bars indicate 95% CI.
(C) (i) Tomographic slices and segmented 3D reconstruction of plasmodesmata in WT and *sac7-1*. Arrows indicate ER-PM detachment. (ii) Contingency plot showing the percentage of plasmodesmata with ER-PM detachment. (iii) Quantification of ER-PM distance in WT and *sac7-1* plasmodesmata at the widest gap. Lines show median. Error bars show 95% CI. Note that WT values in (ii) and (iii) are the same as in Fig 1C (ii) and (iii).
(D) Cell-type dependent dynamic regulation of plasmodesmata MCS and its impact on molecular traffic.

Our results uncover two important findings. First, we reveal that while MCS typically facilitate inter-organellar communication in eukaryotes, they also operate at cell-cell interface to regulate inter-cellular communication. Plasmodesmata, prevalent across the Plantae kingdom, function as unconventional tubular ER-PM MCS. Similar to other MCS, the regulation of membrane contacts at plasmodesmata involves a complex molecular partnership consisting of phosphoinositides and protein tethers. However, unlike typical MCS, governing direct molecular exchange between organelles, plasmodesmata MCS specifically control cell-to-cell molecular flow (Figure 7D). To our knowledge, this marks the first discovery of the functional involvement of MCS in direct intercellular exchange and hints at a new conceptual framework. Recent findings suggest that MCS may also play a role in cell-cell communication in animals. A preprint reports two ER-PM MCS-associated proteins, TMEM24 and C2CD2, to be involved in cell-cell contact in neurons (Johnson et al., 2023), although evidence for effective communication is still lacking. Likewise, Bharathan et al. described close contact between the ER and desmosomal junctions (Bharathan et al., 2023), prompting questions about whether an integrated ER-keratin-desmosome contact zone could transmit ER stress signals between cells. Our study brings this concept one step further by demonstrating the functional involvement of multicellular eucaryotic MCS in direct intercellular exchanges.

The second important outcome of this study proposes a novel regulatory mechanism for cell-cell trafficking. Right now, the textbook model presents callose as the master regulator of plasmodesmata. Unquestionably, numerous studies report the involvement of callose in opening or closing plasmodesmata during various developmental and physiological responses. The prevailing view is that callose buildup creates pressure on the PM, pushing it against the ER and obstructing cellular traffic by reducing the cytoplasmic sleeve (space between the two membranes) size. However, recent research suggests that callose as a plasticizer, enhancing wall flexibility, instead of forming a rigid structure that presses against the PM (Abou-Saleh et al., 2018). Moreover, extreme proximity between the ER and the PM, sometimes as close as 3-4 nm
699 (Nicolas et al., 2017a), likely necessitate membrane bridging-complexes to induce and stabilize
700 this contact. This work suggests that dynamic regulation of plasmodesmata is compromised
701 without the molecular machinery at the ER-PM interface (i.e., PI4P/MCTP), even if callose
702 deposition/removal remains functional. Our data indicate that MCTP/PI4P regulate
703 plasmodesmata largely independent of callose levels. This finding offers a new perspective on
704 how intercellular communication is regulated in plants, and opens the door to a more intricate
705 regulatory mechanism than previously thought.

706 How do MCTP/PI4P dynamically regulate molecular flow across plasmodesmata MCS?
707 The simplest hypothesis is by modulating the ER-PM gap (Figure 7D). Theoretical models
708 suggest that changes in plasmodesmata structure directly affects molecular flow rate (Deinum et
709 al., 2019; Ostermeyer et al., 2022) through resistance to diffusion or convective flow.
710 Overexpression of SAC7 PI4P phosphatase or deletion MCTP3, MCTP4, and MCTP6 tethers
711 increases ER-PM detachment events within plasmodesmata, leading to enhanced molecular flow
712 between cells. This rise in cell-cell trafficking occurs in a context where loss-of-function
713 *mctp3/mctp4* and *mctp3/mctp4/mctp6* mutants also exhibit a reduction of 30-50% of
714 plasmodesmata in the root meristem (Li et al., 2023). This suggests that the transport capacity of
715 individual plasmodesma is considerably greater in *mctp* mutant compared to wild-type.

716 What molecular mechanisms regulate ER-PM MCS? Our data indicate that MCTPs
717 operate in coordination with the anionic lipid PI4P. MCTP4 interacts directly with PI4P through
718 its C2 domains and PI4P's levels directly influence C2s docking to the PM. When PI4P
719 production is up- or down-regulated, cell-cell transport is correspondingly hindered or facilitated.
720 PI4P is a signaling lipid that can vary significantly in concentration depending on the cell types
721 or stress conditions (Noack and Jaillais, 2020). We demonstrated that PI4P phosphatase SAC7
722 regulates MCTP localization and plasmodesmata connectivity in a tissue dependent manner,
723 illustrating that graded PI4P levels can impact cell-cell connectivity in a wild-type context. The
724 SAC7-dependent regulation of MCTP localization occurs at a developmental scale, representing
725 a relatively slow response that is established during tissue differentiation.

726 However, PI4P regulation may also occur on a shorter timescale, within minutes or even
727 seconds so is plasmodesmata regulation during certain biotic or abiotic stresses (Marković and
728 Jaillais, 2022; REF). Some MCTP C2 domains increase lipid-binding upon Ca²⁺ binding (Rizo
729 and Su, 1998; Giordano et al., 2013; Yu et al., 2016), suggesting that variations in Ca²⁺ may
730 rapidly regulate plasmodesmata conductance, through conditional docking. Changes in PI4P or
731 other anionic lipid concentrations might also induce conditional docking of MCTPs to the PM,
732 allowing for rapid opening or closing of the cytoplasmic sleeve. Similar mechanisms were
733 observed in other MCS. For instance, in human cells, PI4P levels at the Golgi dictate ER/Golgi
734 contact establishment and can generate traveling waves of MCS between organelles (Mesmin et
735 al., 2013; Mesmin et al., 2017). Interestingly, in this example, membrane contact establishment is
736 regulated by a PI4P phosphatase called Sac1, which is orthologous to the Arabidopsis SAC7
737 enzyme (Dubois and Jaillais, 2021). Additionally, molecular dynamics suggest that MCTP4 may
738 interact with the PM in different conformations. Whether such conformational changes occur in
739 vivo remains to be determined, but this hypothesis represents an attractive model for regulating
740 cytoplasmic diffusion on a short timescale.

741 Altogether, our study shows that ER/PM tethering not only maintains plasmodesmata structure
742 but also suggests that the ER-PM junction can be dynamically regulated to regulate plant cell
743 connectivity within tissues.

744 **Limitation of the study**

745 A limitation of our work lies in the reliance on loss-of-function *mctp* alleles. The
746 *mctp3/mctp4/mctp6* loss-of-function mutant exhibits structural defects with looser ER-PM
747 contacts, indicating that MCTP tethers not only act as regulatory elements but also as structural
748 elements. One possibility is that callose may not effectively block plasmodesmata due to a
749 significant disruption in plasmodesmata structure in the *mctp* mutant lines. Moreover, we cannot
750 exclude that callose interaction with the PM or other wall-polymer is affected in *mctp* mutant
751 background modifying its action. While we do not expect MCTPs to affect extracellular wall
752 polymers or wall properties directly, we cannot rule out indirect effects. Nevertheless, our data in
753 the *sac7* mutant suggest that increased PI4P levels stabilize MCTP within plasmodesmata and
754 reduces cell-to-cell cytoplasmic diffusion even when callose deposition is inhibited (i.e. after
755 BRZ treatment). While we still do not fully understand the functional and structural relationship
756 between callose deposition and ER/PM tethering within plasmodesmata, our results strongly
757 imply that MCTP/PI4P interaction can close plasmodesmata in the absence of callose.

758 In this study, we focused on the function of PI4P in the regulation of MCTP localization and
759 plasmodesmata activity. However, PI4P is not the only signaling lipid within the PM, which also
760 includes phosphatidic acid, phosphatidylserine and PI(4,5)P₂ (Platre et al., 2018). Because each
761 MCTP protein has multiple C2 domains, which all have the potential to bind to one or several
762 anionic lipids, we need to be cautious and acknowledge that PI4P is likely not the only lipid
763 regulator at plasmodesmata. In fact, we found both PI4P and PI(4,5)P₂ in our purified
764 plasmodesmata fraction. It is possible that each MCTP protein will have specific lipid
765 preferences, thereby regulating ER/PM contact in a differential manner according to the tissue
766 analyzed and/or to environmental stresses. In addition, the interaction between MCTP C2
767 domains and different anionic lipid species may also be modulated by calcium. Altogether, we
768 believe that MCTP/lipid interactions have immense regulatory potential to fine tune
769 plasmodesmata connectivity across a broad range of time scales and with exquisite spatial and
770 temporal specificity.
**Acknowledgments:** We would like to thank Paulo Telles de Souza for sharing Martini3 coarse grain parameter for sitosterol. Fabrice Cordelières for live-imaging data display. Jérôme Hénin for assistance with colvars protocol. Christel Poujol, Jeremie Teillon and Sebastien Marais for assistance with SP8-STED-FLIM and SP5-2P. We would also like to thank Alexandre Martinière, Yohann Boutté, and Sébastien Mongrand for reading and commenting on the article. Light and electron imaging were done at the Bordeaux Imaging Center, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04) or at the RDP lab (ENS Lyon, France). We acknowledge the contribution of the SFR Biosciences (UMS3444/CNRS, US8/Inserm, ENS de Lyon, UCBL) E. Chatre, and J. Brocard at LBIPLATIM-MICROSCOPY for assistance with imaging, and Claire Lionnet for assistance with imaging at the RDP. The lipid analysis was done at the Bordeaux Metabolome platform for lipid analysis (https://www.biomemb.cnrs.fr/en/lipidomic-plateform/) supported by Bordeaux Metabolome Facility-MetaboHUB (grant no. ANR-11-INBS-0010 to LF). We thank Yohann Boutté for his help in anionic lipid extraction, Lothar Kalmbach for the pLOK180 plasmid gift, Yasin Dagdas for the DDRGK1-mCHERRY line and Thomas Eekhout (VIB) for the scRNA seq data mining.

**Funding:** This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (project 772103-BRIDGING to E.M.B. and 101001097-LIPIDEV to Y.J.); the National Agency for Research (Grant ANR-18-CE13-0016 STAYING-TIGHT to E.M.B and Y.J.; ANR-21-CE13-0016-01 DIVCON to A.T. and E.M.B; ANR-18-CE13-0025-02 caLIPSO to Y.J.); the Human Frontier Science Program (project RGP0002/2020, E.M.B.); the French government in the framework of the IdEX Bordeaux University "Investments for the Future" program / GPR Bordeaux Plant Sciences (E.M.B.). Swiss National Science Foundation (project 31003A_179159 to MB. Research Foundation-Flanders grant G002121N (to E.R.) and the HORIZON-MSCA-2023-PF-01 project 101152141-BRACTION (to Y.L.).

**Author contributions:**

Conceptualization: J.P-S., M. S., Y.J., E.M.B

Methodology: J.P-S., M. S., G.D., M.G., S.S., G.D., L.F., F.I., J.T., A.T., Y.J., E.M.B

Validation: J.P-S., M. S., G.D., A.T., Y.J., E.M.B

Investigation: J. P-S, M.S., G.D., M.G., S.S., G.D. V.D., M.P.P., Z. P.L., H.M., A.P., Y.L., L. F., M.S.G., P. C-Q, T. S.M., F.I., V.W., M.D., L.B., P.R., V.B., L.L.L., C.C., S.C., M.Z., Y. L., M.Z.

Supervision: Y.J., E.M.B.

Funding acquisition: A.T., Y.J., E.M.B.

Writing – original draft: J.P-S, M.S., Y.J., E.M.B
810 Writing – review & editing: J.P-S, M.S., G.D., V.D., A.P., P. C-Q, M.P.P., J.T., E.R.

811 M.B., Y.H., A.T., Y.J., E.M.B

812 **Declaration of interests:** The authors declare no competing interests.

813 **Data and materials availability:** All data are available in the main text or the supplementary

814 materials

815

816 **METHODS**

817 • Plant material

818 *Arabidopsis thaliana*, accession Columbia (Col-0), (which we are referring to as wild-type-WT)
819 and transgenic lines (all in Columbia background) were surface sterilized, vernalized at 4C
820 during 2 days and grown vertically on solid half-strength Murashige and Skoog media
821 supplemented with vitamins (2.15 g/L), MES (0.5 g/L), sucrose (10 g/L) and plant agar (7 g/L),
822 pH 5.7 for 6 days at 22°C in a 16-h light/8-h dark cycle with 70% relative humidity and a light
823 intensity of 200 µmol x m⁻² x s⁻¹ prior to use. Recently harvested, synchronized seeds were
824 used for all phenotypical analysis. *Nicotiana benthamiana* plants were grown in an insect proof
825 greenhouse with 18/25°C night/day.

826 The following lines were previously published and characterized *rhd4-1/sac7-1* (EMS) and *rhd4-3/sac7-3* (SALK_079231) (Thole et al., 2008), *EXP7pro::4xYFP* (Marquès-Bueno et al., 2016), *DDRGK1-mCHERRY* (Stephani et al., 2020), *SYT1pro:SYT1-GFP* (Pérez-Sancho et al., 2015b), C4H-GFP (Lee et al., 2019b), RFP-HDEL (Pain et al., 2019), *mctp3-2/mctp4-1* mutant and *MCTP4pro::GFP-MCTP4* (Brault et al., 2019), *35Spro::DRONPA-s* (Gerlitz et al., 2018)

831 • Cell culture

832 *Arabidopsis (Landsberg erecta)* culture cells were cultivated as described in Bayer et al. (Bayer et al., 2004) under constant light (20 µE/m/s) at 22°C. Cells were used for plasmodesmata extraction at 7 days old.

835 • Cloning and plasmid construction

836 In this study, the constructs employed were derived from Col-0 cDNA/genomic DNA, or were
837 synthesized. The promoter sequences were integrated into pDONR-p4RP1, while the genes and
838 fluorescent tags were integrated into either pDONR221 or pDONR-P2RP3, utilizing the
839 MultiSite-Gateway cloning system. For the development of an inducible genome editing vector,
840 plasmids containing the inducible RPS5A promoter, codon-optimized and RFP-tagged Cas9,
841 along with two sgRNAs, were assembled using the Gateway system. The assembly of all
842 constructs was carried out through multi-Gateway reactions, wherein three segments were cloned
843 into the destination vector, pLOK180, which express a red seed-coat selection marker (a gift
844 from L. Kalmbach).

845 The expression vectors were introduced into *Agrobacterium tumefaciens* strain GVG3101, which
846 was then used to transform Arabidopsis Col-0 or *mctp* mutants (as indicated in the figure
847 legends) through the floral dip method, as previously described (Clough and Bent, 1998).
848 Transformed seeds were selected based on the presence of either red seed coat or BASTA
849 resistance, depending on the selection marker used. We employed a similar selection scheme as
850 described in (Lebecq et al., 2022), which ensures that for each transgenic lines selected, several
851 independent transformants, with a single insertion, show similar results. Briefly, between 20 and
852 24 independent T1 (Basta or Green/Red seed coat) were selected and propagated. In T2, all lines
853 were screened using confocal microscopy for fluorescence signal and localization. Between 3
854 and 5 independent lines with a mono-insertion and showing a consistent, representative
855 expression level and localization were selected and grown to the next generation. Each selected
856 line was reanalyzed in T3 by confocal microscopy to confirm the results obtained in T2 and to
857 select homozygous plants. At this stage, we selected one representative line for in-depth analysis
858 of the localization.

859 Cloning of SAC7-related material: The SAC7 promoter (Sac7pro, 1963bp upstream of
860 AT3G51460 START codon) was amplified from Col-0 DNA and inserted in the pENTR-5'-
861 TOPO Gateway vector using TOPO cloning (Invitrogen™). The coding sequence (CDS) of
862 SAC7 (ATG to STOP codon) was amplified by PCR from Col-0 cDNA and recombined by BP
863 reaction into pDONR-P2R-P3. The mCITRINE/pDONR 221, and 2xmCHERRY-
864 1xmyc/pDONR P221 and UBQ10pro/pDONRP4RP1 were published before (Jaillais et al., 2011;
865 Simon et al., 2014). The LR clonase-based three-fragment recombination system (Invitrogen®)
866 was used to produce the pSAC7::mCitrine-SAC7 into pLOK180_pR7m34g (a gift from L.
867 Kalmbach, red seed selection) and pSAC7::2xmCherry-Myc-SAC7; pUBQ10::2xmCHERRY-
868 Myc-SAC7into pLOK292_pFG7m34G (a gift from L. Kalmbach, green seed selection).

869 To generate the inducible *PDR9xve::cals3m*, a 2kb DNA fragment corresponding to *PDR9*
870 promoter, active in the epidermis (Robe et al., 2021) was cloned into p1R4-ML:XVE (Siligato et
871 al., 2016) and subsequently assembled by multi-site gateway technology into the destination
872 plasmid pFG7m34GW (Kalmbach et al., 2023) together with *cals3m* (Vaténet al., 2011) in
873 pDONR221 and the terminator tNOS in pDONRP2R-P3 (Karimi et al., 20007)

874 • Transient expression in *Nicotiana benthamiana*

875 The abaxial side of leaves of 3- to 4-week-old *Nicotiana benthamiana* plants were pressure-
876 infiltrated with GV3101 agrobacterium strains carrying the different constructs. Prior to
877 infiltration, the agrobacterium cultures were grown overnight in Luria and Bertani medium
878 supplemented with rifampicin (50 mg/mL), gentamycin (25 mg/mL), and the construct-specific
879 antibiotic at 28°C. Subsequently, the cultures were diluted to 1/10 in the same growing medium
880 (without Rifampicin) and further cultivated until the optical density at 600 nm (OD600) reached
881 approximately 0.6-0.8. The bacterial cells were then collected through centrifugation at 3000g
882 for 5 min, followed by resuspension in agroinfiltration solution (10 mM MES, pH 5.6, 10 mM MgCl2, and 1 mM acetosyringone) and incubated for 2h at room temperature and in the dark.
883
884 For infiltration of individual constructs, a final OD600 of 0.3 was used. For co-infiltration of two constructs, and OD600 of 0.2 each was used. Additionally, the ectopic silencing suppressor p19 was co-infiltrated in all the cases at an OD600 of 0.05. Post-infiltration, plants were kept in the same growing conditions as before for 2 more days; then leaf-discs were collected from the N. benthamiana leaves and subjected to imaging at room temperature.
885
886
887
888

889 • Lattice light sheet

890 The Lattice Light Sheet Microscope (LLSM) was built according to the technical information
891 provided by the group of E. Betzig at Janelia Research Campus, Howard Hughes Medical
892 Institute (HHMI), USA. The lattice light sheet was focused by a custom excitation objective
893 28.6x 0.66 numerical aperture 3.74-mm (Special Optics). Fluorescence was collected with a CFI
894 Apo LWD 1.1 numerical aperture 25× 2.0-mm detection objective (Nikon) and imaged on a
895 scientific complementary metal-oxide semiconductor (sCMOS) ORCA-Flash4.0 V2 camera
896 (Hamamatsu). The annular mask minimum and maximum numerical apertures were 0.35 and
897 0.4, respectively, thus creating a light sheet with a uniform thickness over length > 30µm. All
898 images were acquired in dithered square lattice mode. We characterized the LLSM optical
899 resolution at l<sub>em</sub>=510nm using 170nm diameter beads. We found 275 +/- 5 nm and 690 +/- 10
900 nm, laterally and axially respectively. Images were acquired at ~10 to 20 µm below the surface
901 of the root. Fast sample translation with a piezo stage was used to acquire Z stacks with a step of
902 300 nm. Typical laser power incident on the excitation objective is ~ 0.4mW. The raw data are
903 3D deconvolved with an open-source Richardson Lucy (RL) algorithm
904 (https://github.com/dmilkie/cudaDecon) using an experimental PSF. The RL software is bundled
905 into LLSpy, a python LLSM data processing and visualization toolbox
906 (https://github.com/tlambert03/LLSpy). Deconvolution runs on a CUDA compatible GPU
907 graphics card. A 3x3 median filter (noisy pixel removal) followed by background subtraction is
908 applied before RL deconvolution with 10 iterations.
909

909 • Laser Scanning Confocal Microscopy

910 In the conducted live-imaging experiments, a Zeiss LSM 880 confocal microscope equipped
911 with 40x and 63x oil-immersion objectives, as well as 40x water immersion objectives, was
912 employed. The microscope was controlled using ZEN Black 2011 software. The experiments
913 were carried out at a controlled temperature ranging from 22 to 24 C.

914 To detect GFP in the confocal mode, a filter with an excitation (Ex) wavelength of 488 nm and
915 an emission (Em) range of 505-550 nm was utilized. For the detection of tagRFP or propidium
916 iodide (PI) fluorescence, a filter with an Ex wavelength of 556 nm and an Em range of 570-625
917 nm was employed. To detect YFP fluorescence, a filter with an excitation (Ex) wavelength of
918 514 nm and an emission (Em) range of 520-570 nm was utilized when imaging YFP alone.
919 When YFP was imaged together with a red reporter, we used the settings already described for
920 GFP.

921 For Airyscan imaging, GFP or YFP fluorescence was detected using an excitation (EX)
922 wavelength of 488 nm and an emission bandpass (Em BP) range of 420-480 nm in combination
923 with 495-550 nm. On the other hand, for the detection of PI an excitation (EX) wavelength of
924 488 nm and an emission (Em) range of 620-700 nm in combination with a long pass (LP) filter at
925 645 nm were used. For callose imaging, atto550 excitation was done with 561 nm power
926 excitation and fluorescence collected between 566 and 700 nm. Callose deposition at
927 plasmodesmata was quantified with Fiji software and signal from the forming cell plae was
928 excluded as indicated in Figure 3. Calcofluor aquisition was done by 405nm excitation and
929 collected at 410-480nm. For SAC7/MCTP colocalization (Figure 6F), Airyscan imaging was
930 conducted on 7-day-old roots using an inverted LSM 980 Axio Observer Z1/7 (Carl Zeiss group,
931 http://www.zeiss.com/) equiped with an AiryScan 2 module, GaAsP-PMT detectors and a C
932 Plan-Apochromat 40X objective (numerical aperture 1.3, oil immersion).

933 To prevent signal cross-talk, image acquisition occurred by frame when two colors were imaged
934 simultaneously.

935 • Spinning Disk Microscopy:

936 Spinning disk microscopy was used in Figure 6A-D and 7A. Observations of roots of 7-day-old
937 seedlings were conducted using a Zeiss microscope (AxioObserver Z1, Carl Zeiss Group,
938 http://www.zeiss.com/) equipped with a spinning disk module (CSU-W1-T3, Yokogawa,
939 www.yokogawa.com) and a Prime 95B camera (Photometrics, https://www.photometrics.com/).
940 We used a 40x objective (Plan-Apochromat, numerical aperture 1.1, water immersion) in Figure
941 7A or a 63x objective in Figure 6A-D (Plan-Apochromat, numerical aperture 1.4, oil immersion).
942 Green Fluorescent Protein (GFP) was excited with a 488 nm laser (150mW), and the ensuing
943 fluorescence emission was filtered via a 525/50 nm BrightLine® single-band bandpass filter
944 (Semrock, http://www.semrock.com/). The mCITRINE marker was excited with a 515nm laser
945 (60mW), and the resulting fluorescence emission was directed through a 578/105 nm
946 BrightLine® single-band bandpass filter (Semrock, http://www.semrock.com/). For mCHERRY
947 or PI labeling, an excitation laser at 561nm (80mW) was applied, and the fluorescence emission
948 was filtered using a 609/54 nm BrightLine® single-band bandpass filter (Semrock,
949 http://www.semrock.com/).

950 • Cryofixation and freeze-substitution

951 Five-day old seedlings of WT, *mctp3/4* and *mctp3/4/6* were grown vertically MS ½. Roots were
952 taken and cryofixed in 20% BSA filled copper platelets (100 nm deep and 1.5 mm wide) with
953 EM PACT1 high-pressure freezer (Leica). Subsequently, the root tissues were harvested and
954 subjected to cryopreservation by rapidly freezing them using a high-pressure freezer (Leica EM
PACT1) equipped with copper platelets (100 nm deep and 1.5 mm wide) filled with 20% bovine serum albumin (BSA) solution. The cryofixed samples were then subjected to freeze-substitution within an AFS2 system (Leica) maintained at -90 °C. The cryosubstitution process involved immersing the samples in a cryosubstitution mixture containing 0.36% uranyl acetate in pure acetone for a duration of 24 hours. Subsequently, the temperature was gradually increased at a rate of 3 °C per hour until it reached -50 °C and held steady for 3 hours. To ensure thorough substitution, the cryosubstitution mixture was replaced with pure acetone and later pure ethanol, with each solvent being applied for three 10-minute washes. The copper platelets were intentionally retained to prevent any potential loss of the samples. Infiltration of the specimens was carried out using a series of increasing concentrations of HM20 Lowicryl resin (Electron Microscopy Science), including 25% and 50% (1 hour each), 75% (2 hours), and finally 100% (overnight, 4 hours, 48 hours, with fresh resin for each bath). Following infiltration, the samples were polymerized under ultraviolet light for 24 hours at -50 °C, after which the temperature was incrementally raised at a rate of 3 °C per hour until it reached 20 °C, where it was maintained for an additional 6 hours.

970 • Tomography and Transmission Electron Microscopy

971 Fixed root embedded in HM20 resin blocks were sectioned longitudinally into slices with a thickness of 80 nm using an EM UC7 ultramicrotome (Leica). These sections were then 972 delicately positioned onto 200 mesh copper grids. Subsequent observations were conducted 973 employing a FEI TECNAI Spirit 120 kV electron microscope. For plasmodesmata structure we 974 acquired tomograms in 3 to 4 roots over two technical replicates. 975

976 For the purposes of tomographic analysis, a protocol in accordance with the approach outlined 977 by Nicolas et al., 2017a was employed. To facilitate image alignment during the tomography 978 process, sections were coated with 5 nm colloidal fiducials. A series of tilt images spanning an 979 angular range from -65° to 65° was acquired with a 1° angular increment using the FEI 3D 980 explore tomography software. Subsequently, tomographic reconstructions were generated 981 utilizing the eTomo software (http://bio3d.colorado.edu/imod/). Segmentation tasks were carried 982 out employing the 3dMOD software (https://bio3d.colorado.edu/imod/doc/3dmodguide.html).

983 • Forester Resonance Energy Transfer-Fluorescence Lifetime Imaging (FRET-FLIM)

984 The confocal microscope used for FRET FLIM was a Leica SP8 WLL2 on an inverted stand 985 DMI6000 (Leica Microsystems, Mannheim, Germany), using objectives HC Plan Apo CS2 40X 986 oil NA 1.30. The confocal microscope was equipped of a pulsed white light laser 2 (WLL2) with 987 freely tuneable excitation from 470 to 670 nm (1 nm steps) and a pulsed diode at 440 nm and 988 also a diode laser at 405 nm.

989 The scanning was done using either a conventional scanner (10Hz to 1800 Hz) or a resonant 990 scanner (8000Hz). The microscope was composed of 2 internal photomultiplier tubes (PMT), 2
991 internal hybrid detectors and 1 external PMT for transmission. For excitation of the donor
992 *pMCTP4::e-YFP*- MCTP4, 516nm laser was used. The confocal microscope was equipped with
993 the FALCON module for FLIM measurements. We used 5 iterations in order to acquire enough
994 photon counts (10-4). Monoexponential fit was applied for both donor alone and donor with the
995 acceptor (*pMCTP3::mCherry* MCTP3) and values were extracted only from the plasmodesmata
996 signal.

997 • Two-photon microscopy and cell diffusion assay with DRONPA-s

998 5-day-old 35S::DRONPA-s seedlings was cultivated on ½ MS plates under long-day conditions
999 at 21 °C with 70% humidity. Subsequently, these seedlings were transferred to either DMSO or
1000 200nM Brassinolide plates for an additional 24 hours. The root tips of the seedlings were
1001 subjected to analysis using a Leica TCS SP5-Multi photon confocal microscope setup. Prior to
1002 imaging, the plants underwent a 2-minute incubation in a 1/500 propidium iodide (PI) solution in
1003 water. PI was excited at 488 nm and its emissions were detected at 620-700 nm. Leica LAS AF
1004 software was employed for the quantification of fluorescence intensities.

1005 The movement of DRONPA-s was captured as follows:

1006 1. DRONPA-s deactivation: Root tips were illuminated with 488 nm light for 45 seconds at
1007 70% of the total laser intensity using an argon laser (20mW, Leica Microsystems).

1008 2. DRONPA-s activation: Three Regions of Interest (ROIs), each corresponding to a single
1009 cell per root, were selected at the cell center to avoid activating adjacent cells. These
1010 ROIs were exposed to 5 seconds of 800 nm light using a two-photon IR (Titane-Saphir
1011 pulsating laser). The laser power used was approximately 2.6W with a 20% gain.

1012 3. DRONPA-s acquisition: Detection was conducted 2 seconds after the single cell
1013 activation and continued for 120 seconds, using a 20% 488 nm argon laser at an emission
1014 range of 500-575 nm.

1015 DRONPA-s movement analysis involved measuring mean fluorescence intensities from the
1016 activated cell and the two adjacent cells using ImageJ. DRONPA movement was calculated
1017 based on the fluorescent signal transferred from the activated cell to the adjacent cells. Any X-Y
1018 drifts were corrected with the StackReg plugin. Following normalization and background
1019 subtraction, the values were converted into a percentage of the average fluorescence from the
1020 two adjacent cells. The activated cell was considered to have 100% DRONPA-s molecules
1021 activated in the ROI. A total of more than 20 roots were analyzed, with 2-3 ROIs per root.

1022 • Fluorescence Recovery After Photobleaching

1023 PI permeability assessments were made using FRAP on six day-old Arabidopsis root tips co-
1024 stained with CFDA (50µg/mL) and Propidium iodide. Roots were incubated in an aqueous CFDA
1025 solution for 5 minutes, then successively washed out in 3 water baths and mounted with
propidium iodide in water for imaging FRAP experiments were conducted using a Zeiss LSM 880 confocal microscope, which was equipped with a Zeiss CPL APO x 40 oil-immersion objective (numerical aperture 1.3). To excite CFDA, a 488 nm wavelength was utilized with 100% of argon laser power, and the fluorescence emission was collected within the 505–550 nm range using a GaAsp detector. Photobleaching was performed on rectangular regions of interest (ROIs) encompassing three epidermal cells, with the exciting laser wavelengths set to 100%. The FRAP procedure involved capturing 10 pre-bleach images, followed by 15 iterations of bleaching, with a pixel dwell time of 1.31 µs. Subsequently, 90 post-bleach images were acquired. The recovery profiles were subjected to background subtraction and then double normalization through the FRAP analysis website https://easyfrap.vmnet.upatras.gr/ (Koulouras et al., 2018).

• Quantification of the Apico Basal Vs Intracellular Fluorescence Ratio (APFIR):

Two methods were used to obtain the APFIR, a “manual” approach with a draw-by-hand fluorescence extraction ROI method on Fiji (Figure 6) and an “automated” pipeline with a Fiji macro (Figure 7). In figure 6, we could not use the automated approach because the signals from the various ER markers were too different (perinuclear and diffuse vs subcortical) leading to error in the segmentation.

Fluorescence extraction:

**Manual method:** the intracellular fluorescence was extracted on each cells by drawing an ROI close to the nucleus to capture most of the perinuclear ER, the apico-basal fluorescence was extracted by drawing two ROI at the apical and basal membranes of each cells, the background fluorescence was extracted by drawing an ROI inside the nucleus.

**Automated method:** This method requires a segmentation of all the cells, which is done with the red channel (propidium iodine staining of the root). Subsequently to segmentation, the fluorescence of interest (green channel) is extracted at different ROI automatically determined by the macro for each cells: the intracellular fluorescence (the entire fluorescence inside the cell); the apical membrane fluorescence, and the basal membrane fluorescence. The macro can be found at:

https://github.com/RDP-vbayle/SiCE_FIJI_Macro/blob/main/misc/MacroAPFIR_Perez-Sancho et al_221020.ijm

Calculation of the APFIR:

No background subtraction is done on the ROI fluorescence values obtained via the automated method. For the fluorescence values obtained via the manual method, the values of the ROI fluorescence (apical, basal, intracellular) is subtracted by the value of the background of the
1061 same cell. For each cell, the APFIR is calculated as the mean apico-basal fluorescence divided
1062 by the intracellular fluorescence.

1063 • Co-Immunoprecipitation in *Nicotiana benthamiana*.

1064 The appropriate constructs were transiently expressed in Nicotiana benthamiana (see Transient
1065 expression in *Nicotiana benthamiana* section). Two plants per construct combination and two
1066 whole leaves per plant were agroinfiltrated. Using a puncher, 10 leaf-discs were collected for
1067 each leaf, so 40 leaf-discs per construct combination, and immediately frozen in liquid nitrogen
1068 (aprox 0.5 g of tissue). Frozen tissue was grinded using mortar and pestle and total proteins were
1069 extracted with 1 mL of extraction buffer (150 mM Tris-HCl, pH 7.5; 150 mM NaCl; 10 %
1070 glycerol; 10 mM EDTA, pH 8; 1mM NaF; 1 mM Na2MoO4; 10 mM DTT; 0.5 mM PMSF; 1%
1071 (v/v) P9599 protease inhibitor cocktail (Sigma); 1 % (v/v) Igepal) by incubating during 40 min at
1072 4 C with continuous mixing in an end-over-end rocker. Samples were centrifuged for 20 min at
1073 4C and 9,000 g. Supernatants were filtered by gravity through Poly-PrepChromatography
1074 Columns (#731-1550 Bio-Rad). For each sample, 50 uL of the filtered supernatant were
1075 collected and mixed with 50 uL of Laemmli buffer (Tris-HCl pH 6.8 125 mM; 4% SDS; 20 %
1076 (v/v) glycerol; 2 % (v/v) beta-mercaptoethanol; 0.01 % bromophenol blue) for immunoblot
1077 analysis as input. The remaining filtered supernatants were mixed in a 1:1 proportion with wash
1078 buffer (extraction buffer without Igepal) to reach a final concentration of 0.5 % Igepal in the
1079 samples before adding the GFP-Trap agarose beads (as recommended by the manufacturer to
1080 avoid nonspecific binding). 15 uL of equilibrated GFP-Trap agarose beads were added to each
1081 sample and incubated during 2 h at 4 C with continuous mixing. After incubation, samples were
1082 centrifuged 30 s at 500 g to precipitate the beads Then beads were washed 3 times with a wash
1083 buffer. After the third wash, a final centrifugation during 30 s at 2000 g was performed and the
1084 supernatant was discarded. Finally, immunoprecipitated proteins were eluted by incubating the
1085 beads with 50 uL of Laemmli buffer at 70 C for 20 min followed by 2 min centrifugation at 2500
g and recovery of the supernatant. Input and immunoprecipitated samples were analyzed by
1086 western blot.
1087

1088 • Lipid inhibitor treatments

1089 PAO treatment was used to inhibit PI4kinases (Simon et al., 2016b). 25-30 minutes of 60µM
1090 PAO was used to 5 days old p35S::DRONPA-s in WT and mctp4/3 mutants background then
1091 imaged. Wortmanin was used to inhibit PI3 kinase (Gomez et al., 2022) at a concentration of
1092 10µM for 40 minutes and YM 201653 was used to inhibit PI3,5kinase at a concentration of 2µM
1093 and treated for 1 hour.

1094 • Environmental and hormonal triggers with brasinosteroids and NaCL

1095 For Brasinolide (BL) and Brassinazole (BRZ) treatment 4 days old roots of *p35S::DRONPA-s* in
1096 WT and mctp4/3 background were transferred in ½ MS plate containing 200nM BL or 1µM
1097 BRZ and grown for 24h before imaging. DMSO was used as a mock solution/ Salt treatment was
1098 performed by growing the same lines for 5 days and 20mM NaCl was applied 15 minutes before
1099 imaging. Water was used as a control solution.

1100 • Callose immunostaining

1101 Arabidopsis seedlings were vertically cultivated on ½MS agar plates for a duration of 4 days.
1102 Subsequently, they were transferred to fresh media and subjected to various treatments for an
1103 additional 24 hours. The treatments included 200 nM BR, 1 µM BRZ, DMSO mock, and a 15-
1104 minute exposure to 20mM NaCl.

1105 The immunolocalization procedure was performed following a previously published protocol
1106 (Pendle and Benitez-Alfonso, 2015). All solutions were prepared in microtubule stabilization
1107 buffer (MTSB), comprising 50 mM PIPES, 5 mM EGTA, 5 mM MgSO4, and adjusted to pH 7
1108 with KOH. Seedlings were fixed in 4% (v/v) paraformaldehyde for 1h followed by 4 washes
1109 with MTSB. Root tips were then excised and mounted on microscopy slides coated with poly-
1110 lysine.

1111 Immunolabelling was performed using the Immunorobot InSituPro VSI from Protigene. Briefly,
1112 the procedure includes cell-wall permeabilization with driselase 2% during 15 min and plasma
1113 membrane permeabilization by 10 % DMSO and 3 % Igepal during 1 h. To prevent nonspecific
1114 binding, the samples were subjected to a blocking step using 5% neutral donkey serum before
1115 incubation with the primary antibody. The callose antibody (obtained from Australia
1116 Biosupplies) was appropriately diluted to 1/500 in MTSB supplemented with 5% (v/v) neutral
1117 donkey serum and incubated with the samples at room temperature for 4 hours.

1118 For the subsequent step, the secondary anti-mouse antibody, coupled to atto550 (Merk) or alexa
1119 fluor 594, was diluted to 1/1000 in MTSB buffer containing 5% (v/v) neutral donkey serum. This
1120 antibody solution was then applied to the samples and allowed to incubate for 1 hour.

1121 • Root phenotyping

1122 *Phenotyping of the root growth rate* (Schindelin et al., 2012; Slovak et al., 2014; Platre et al., 2023).
1123 Wild-type, single (*mctp3-2*, *mctp3-1*, *mctp4-1*, *mctp4-2*, *mctp6-1*), double (*mctp3-2/mctp4-1*,
1124 *mctp3-1/mctp4-1*, *mctp3-2/mctp6-1*) and triple (*mctp3-1/mctp4-1/mctp6-1*) mutant seeds were
1125 sowed in 12-cm x 12-cm square plates filled with 1/2 MS medium containing 0.8% of agar and
1126 0.5% of sucrose and were stratified for 2-3 days at 4°C before being transfer to a growth
1127 chamber. Six days after planting, about 20 seedlings were transferred using forceps to an
1128 imaging chamber (Lab-Tek, Chambers #1.0 Borosilicate Coverglass System, catalog number:
1129 155361) filled with the identical medium described above. Note that the transfer took about 45-
1130 60 seconds. Images were acquired every minute for 6 hours, in brightfield conditions using a
1131 Keyence® microscope model BZ-X810 with a BZ NIKON Objective Lens 2X CFI Plan-Apo
Lambda (Platre et al., 2021). The same plants were then transferred to 12-cm x 12-cm square plates filled with 1/2 MS medium containing 0.8% of agar and 0.5% of sucrose and grown to three more days and scanned to calculate the root growth rate per day.

*Quantification of the root growth rate.* For time lapse analysis of the root growth rate the script was used "SCRIPT_MCTP" and is available on https://github.com/mplatre/SCRIPT_MCTP.git.
To calculate the root growth rate per day the plates were scanned using BRAT software (Slovak et al., 2014). We then calculated the root length for three days and divided by three to evaluate the root growth rate per day using Fiji (Schindelin et al., 2012).

*Root hair characterization and complementation analysis.* For quantitative root hair analysis, seedling were grown in vitro, in a RH-media (2.15 g/L of MS (Duchefa, "0.5 MS"), 20 g/L of sucrose, 100 mg/L of myo-inositol, 0.5 mg/L of nicotinic acid, 0.5 mg/L of Vitamin B6, 0.1 mg/L of Vitamin B1, 2 mg/L of Glycine, 6 g/L Phytagel (Sigma-Aldrich)). Root hair length was assessed in 5-day-old seedlings, specifically focusing on homozygous T3 lines. Root hairs falling within the focal plane, spanning from 2000 µm to 4000 µm distance from the root apex, were subjected to measurement. A total of around 400 to 600 root hairs were measured for each transgenic line across two separate repetitions (approximately 200 to 300 root hairs per repetition, encompassing roughly 10 roots per repetition).

* Liposome binding assays

Experiment was performed as in Pérez-Sancho et al. (Pérez-sancho et al., 2016), with small modifications:

*Protein production and purification:* The C2B domain of MCTP4 was expressed as His-tagged protein using the expression vector pET28 and transformed in E. Coli BL21 (DE3). A 5 mL culture of transformed bacteria in LB medium with 50 µg/mL of Kanamycin (Kan) was grown overnight at 37 °C with continuous rotation at 200 rpm. 50 mL LB medium + Kan was inoculated with 2 mL of the overnight grown culture and incubated at 37 °C for 2-3 h, until the OD600 was between 0.6 and 0.8. Then, the expression of MCTP4-C2B was induced by adding 100µM IPTG to the culture and incubating overnight at 18 °C. A sample of the culture before and after protein induction was collected to assay protein production by SDS-PAGE. Cell culture was centrifuged at 1600 g during 15 min and the pellet (1 g) was resuspended in 3 mL of buffer A (HEPES NaOH 50 mM pH 7.6, NaCl 150 mM, Glycerol 10%, DTT 1 mM). Cells were disrupted by sonication (three cycles of 2 min, with 20 s on and 40 s off and 60 % amplitude, keeping the tube on ice) and the suspension was centrifuged at 100 000 g and 4 °C for 30 min. The supernatant was collected, mixed with 600 µL of Ni-NTA beads slurry (300 µL beads) and 125 µL of Benzoase (250 U/ µL), and incubated for 1 h at 4°C with rotation to allow proteins to bind to the beads. The mixture was centrifuged at 5000 g and 4 °C for 5 min and the pelleted beads were washed 5 times with 1.5 mL of buffer A. Finally, proteins were eluted with buffer A
1168 containing 500 mM imidazole and their concentration was determined by Nanodrop (Mw 21278
1169 and Σ 32430).

1170 Liposome preparation: Liposomes of different composition were prepared by mixing the
1171 corresponding lipids stocks in chloroform. Lipid stocks were: PC at 10 mg/mL, PS at 10 mg/mL,
1172 PI(4)P at 5 mg/mL, PA at 10 mg/mL, PI(4,5)P2 at 5 mg/mL, and DOPE-Rho (1,2-dioleoyl-sn-
1173 glycero-3-phosphoethanolamine conjugated to rhodamine) at 1 mg/mL (used to visualize the
1174 lipid fraction during the preparation); and were mixed as indicated in the table below:

<table><thead><tr><th></th><th>PC (µL)</th><th>PS (µL)</th><th>PI(4)P (µL)</th><th>PA (µL)</th><th>PI(4,5)P2 (µL)</th><th>DOPE-Rho (µL)</th></tr></thead><tbody><tr><td>PC</td><td>78</td><td>0</td><td>0</td><td>0</td><td>0</td><td>13</td></tr><tr><td>PC:PS</td><td>62</td><td>20</td><td>0</td><td>0</td><td>0</td><td>13</td></tr><tr><td>PC:PS:PI(4)P</td><td>42</td><td>20</td><td>40</td><td>0</td><td>0</td><td>13</td></tr><tr><td>PC:PS:PA</td><td>50</td><td>20</td><td>0</td><td>7</td><td>0</td><td>13</td></tr><tr><td>PC:PS:PI(4,5)P2</td><td>42</td><td>20</td><td>0</td><td>0</td><td>44</td><td>13</td></tr></tbody></table>

1176 Lipid mixtures were dried as a thin layer using a nitrogen stream and the lipid layers were
1177 resuspended in 1 mL of buffer A by vortexing at room temperature during 20 min. Then, they
1178 were subjected to 5 cycles of freezing in liquid nitrogen and thawing in a water bath at 37 °C and
1179 extruded 10 times through an 800 nm pore size polycarbonate membrane.

1181 Liposome binding and sedimentation: 10 µg of purified MCTP4-C2B and 40 µL of liposomes
1182 were mixed in buffer A containing 10 mM CaCl2 (1 mix for each different liposome
1183 composition) until a final volume of 200 µL and incubated at room temperature for 15 min
1184 without agitation. Large multilamellar vesicles (and the proteins bound to them) were collected
1185 by centrifugation at 300 000 g and 4°C for 45 min. 195 µL of supernatants were recovered and
1186 the pellets were resuspended in 195 µL of buffer A. Supernatant and resuspended-pellet fractions
1187 were mix with Coomassie blue buffer and run into SDS-PAGE for protein separation and
1188 quantification. Only one band corresponding to the C2B domain of MCTP4 was observed for
1189 each of the samples and each of the repetitions of the experiment. Gels were imaged using a Bio-
1190 rad's stain-free imager on TGX-Stain-Free acrylamide gels and proteins quantified using Fiji.

1192 • Plasmodesmata purification and lipid analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS)
1193
1194

1195 The cell wall fractions were prepared from 6-day-old Arabidopsis suspension cultured cells,
1196 following the methodology outlined by Bayer et al. (Bayer et al., 2004). However, in this study,
1197 the cell disruption process was repeated five consecutive times using the N2 cell disruption
1198 vessel, in contrast to the standard three repetitions. To obtain the plasmodesmata-enriched
1199 membrane fraction, purified wall fragments were utilized, employing the method described by
1200 (Fouillen et al., 2022). Briefly, purified cell walls were subjected to digestion with 0.7% (w/v)
1201 cellulase R10 (Karlan) in a digestion buffer comprising 10 mM MES (pH 5.5) and 4.4%
1202 mannitol, supplemented with 1 µM phenylmethylsulfonyl fluoride and a complete protease
1203 inhibitor cocktail (Roche Diagnostic). The digestion process was carried out at 37°C for 1.5
1204 hours with continuous shaking at 50 to 100 rpm. Following the digestion step, centrifugation was
1205 performed at 5850g for 5 minutes at 4°C to separate the supernatant and pellet fractions. The
1206 pellet underwent additional washing in an excess volume of digestion buffer before undergoing a
1207 second centrifugation step. The two supernatants were combined and subjected to further
1208 centrifugation at 110,000g for 40 minutes at 4°C. The resulting pellet, which contained
1209 plasmodesmata-derived membranes, was washed with an excess volume of cold Tris-buffered
1210 saline (TBS; 20 mM Tris-HCl, 0.14 M NaCl, and 2.5 mM KCl, pH 7.4). Finally, the pellet was
1211 resuspended in cold 1× TBS containing protease inhibitors (Roche Diagnostic). It is noteworthy
1212 that approximately 600 mL of cultured cells was employed to yield 100 µg of plasmodesmata-
1213 enriched membrane fraction. The protein amount in the samples was determined using a
1214 bicinchoninic acid protein assay with BSA as the standard. For the purification of
1215 plasmodesmata-enriched membrane fractions from cells subjected to sterol inhibitor treatment,
1216 fen was added to 5-day-old liquid cultured Arabidopsis cells at a final concentration of 250
1217 µg/mL (stock solution 500 µg/µL in DMSO) and incubated as previously described for 48 hours
1218 before the purification process.

1219 For anionic lipid analysis we followed the protocol described in (Genva et al., 2023). In brief,
1220 plasmodesmata fraction was incubated with 725 µL of a MeOH/CHCl3/1M HCl (2/1/0.1 v/v/v)
1221 solution and 150 µL water in presence of 10 ng of 17:0-20:4 PI(4)P standard (Avanti). 750 µl of
1222 chloroform and 170 µl HCl 2M were then added. The samples were vigorously shaken and
1223 centrifuged. The lower phase was washed with 700 µl of the upper phase of a mix of
1224 methanol:chloroform:HCl 0.01M 1:2:0.75, vortexed and centrifuged. Then, samples were kept
1225 overnight at -20 °C.

1226 The organic phase was transferred to a new Eppendorf and the methylation reaction was carried
1227 out. For this purpose, 50 µL of TMS-diazomethane (2M in hexane) were added to each sample.
1228 After 10 min, the reaction was stopped by adding 6 µL of glacial acetic acid. 700 µL of the upper
1229 phase of a mix of MeOH/CHCl3/H2O (1/2/0.75 v/v/v) was added to each sample which was then
1230 vortexed and centrifuged (1500 x g, 3 min). The upper phase was removed and the washing step
1231 was repeated once. Finally, the lower organic phases were transferred to a new Eppendorf.
1232 Following the addition of 100 µL MeOH/H2O (9:1 v/v), the samples were concentrated under a
1233 gentle flow of air until only a drop remained. 80 µL of methanol were added to the samples,
1234 which were submitted to ultrasounds for 1 minute, before adding 20 µL water, and were
1235 submitted to 1 more-minute ultrasound. The samples were finally transferred to HPLC vials for
1236 analysis.
Analysis of methylated anionic phospholipids (Genva et al., 2023) were performed using a liquid chromatography system (1290 Infinity II, Agilent) coupled to a QTRAP 6500 mass spectrometer (ABSciex). The chromatographic separation of anionic phospholipid species was performed on a reverse phase C18 column (SUPELCOSIL ABZ PLUS; 10 cm x 2.1 mm, 3µm, Merck) using methanol/water (3/2) as solvent A and isopropanol/methanol (4/1) as solvent B at a flow rate of 0.2 mL/min. All solvents are supplemented with 0.1% HCCOOH and 10 mM ammonium formate. 10 µL of samples were injected and the percentage of solvent B during the gradient elution was the following: 0-20 min, 45%; 40 min, 60%; 50 min, 80%. The column temperature was kept at 40 °C. Mass spectrometry analysis was performed in the positive ionization mode. Mass spectrometry data were treated using the MultiQuant software (ABSciex). Nitrogen was used for the curtain gas (set to 35), gas 1 (set to 40), and gas 2 (set to 40). Needle voltage was at +5500 V with needle heating at 350 °C; the declustering potential was +10 V. The collision gas was also nitrogen; it was set between 26 to 45 eV according to the lipid classes.

* Identification of peptides by mass spectrometry.

Each sample was deposited on a 10% acrylamide SDS-PAGE. After a short migration, gels were stained with colloidal blue overnight. The bands of interest from the SDS-PAGE gel were cut and destained. Reduction of disulfide bonds and alkylation of cysteine were performed, and proteins were finally digested overnight with trypsin. Resulting peptides were extracted from the gel, acidified and were injected on a Ultimate 3000 nanoLC system (Dionex, Amsterdam, The Netherlands) coupled to a Electrospray Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA). The spectra obtained were analyzed by Proteome Discoverer 3.1 with Sequest HT as the search algorithm, using an *Arabidopsis thaliana* protein database (Araport_20220103; 40,668 entries), including 2 missed cleavage sites by trypsin, carbamidomethylation of cysteine (+57 Da) as static modification) and oxidation of methionine (+16 Da), acetylation of protein N-term (+42 Da), Methionine loss (-131 Da) methionine loss/acetylation (+89 Da) as dynamic modification. Peptide validation was performed using Percolator algorithm and only "high confidence" peptides were retained corresponding to a 1% False Positive Rate at peptide level. Peaks were detected and integrated using the Minora algorithm embedded in Proteome Discoverer.

Protein abundances were calculated as the sum of the abundance of corresponding unique peptides. Normalization was performed based on total *Arabidopsis thaliana* protein amount. Protein ratios were calculated as the median of all possible pairwise peptide ratios. A t-test was calculated based on background population of peptides or proteins. Quantitative data were considered for proteins quantified by a minimum of two peptides and a statistical p-value lower than 0.05.

* Molecular dynamics
1273 *Membrane Creation:* We used the Insane program (Jumper et al., 2021) to generate three
1274 membranes with a composition representative of the plasma membrane: (70%,68%,64%)
1275 phosphatidylcholine (PC), 20% Sitosterol, 10% phosphatidylserine (PS), (0%, 2%, 6%) PI(4)P.
1276 Two lipids present in our modeled membrane were not present in the Martini 3 topology library:
1277 PS of plants that have a longer tail (18:2 24:1) and sitosterol. The topology for PS (18:2 24:1)
1278 was created by analogy, using the topology files of PIPS and DNPS. The Sitosterol model was
1279 obtained from Paulo C. T. Souza and Luís Borges Araújo following the recent parameterization
1280 of cholesterol in MARTINI 3 (Borges-Araújo et al., 2023).

1281 *Protein Setup:* Three prediction tools, AlphaFold Monomer (Jumper et al., 2021), AlphaFold
1282 Multimer (Souza et al., 2021), and RosettaFold (Baek et al., 2021), were employed to determine the
1283 3D structure of MCTP4. These methods converged on a single solution for the C2 domains.

1284 All simulations were conducted using Gromacs 2021.5
1285 (https://manual.gromacs.org/2021.5/release-notes/index.html), supplemented with the Colvar
1286 plugin (Fiorin et al., 2013). The Martini 3 force field (Souza et al., 2021) was utilized. We applied
1287 a constraint network to the protein to stabilize the 3D structure of the cytoplasmic domain. The
1288 protein's center of mass was positioned 10 nm from the membrane. We replicated each condition
1289 five times. Each system underwent minimization and equilibration at 300K. The protocol
1290 included an initial phase of energy minimization, followed by multi-stage equilibration, and
1291 concluded with a production simulation. Energy minimization was performed in two iterations of
1292 5,000 steps each, using the steepest descent method. The simulations were then equilibrated in
1293 five stages, using time steps of 2, 5, 10, 15, and 20 fs. A target temperature of 300 K was
1294 maintained using the v-rescale thermostat, with a coupling constant of 1 ps. A semi-isotropic
1295 pressure of 1 bar was maintained using the Parrinello-Rahman barostat, with a compressibility of
1296 $4.5 \times 10^{-5}$ bar$^{-1}$ and a relaxation time constant of 12 ps. Long-range interactions were handled
1297 with a cutoff radius of 1.1 nm for both van der Waals and Coulombic interactions, implementing
1298 a switching function from 1.0 nm for van der Waals. The production simulations were executed
1299 in an NPT ensemble with a time step of 20 fs for a total simulation time of 3 microseconds.
1300

1301 *Simulation Condition*
1302
1303 - Colvars

1304 Two collective variables (colvars) were used to maintain a realistic orientation of the protein
1305 relative to the membrane. The colvar tilt was chosen to control the protein's tilt angle, and the
1306 colvar distanceZ was implemented to prevent the protein from passing through the periodic box.
1307

1308 - Protein Variants
1309 Specific mutations were introduced to study their impact on the functionality and interaction of
1310 proteins with membranes. These mutations were identified by analyzing contacts in our
1311 simulations with PI4P and using a prediction tool for membrane-interacting residues,
1312 hydrophobic protrusions (https://reuter-group.github.io/peprmint/pepr2vis).

1313 The following mutations were explored:

1314 Five residues on the C2C domain (D217N, K218Q, R220Q, K318Q, K322Q).

1315 To observe the effect of the presence or absence of PI4P on the interaction of the protein with the

1316 membrane we performed simulations with various PI4P concentrations (without PI4P or with a

1317 2% concentration of PI4P). Then to assess the impact of these modifications on the domain of

1318 the protein, we perform simulations C2C variant. In each case, all three domains of the protein

1319 were retained in the mutants to ensure a comprehensive analysis.

1320
1321 - Calculation of Minimum Distances
1322

1323 To evaluate interactions between mutated proteins and PI4P molecules, the minimum distance
1324 between the mutated regions of the proteins and PI4P was calculated using the gmx mindist tool
1325 from GROMACS. In the condition without PI4P we compute the distance with the membrane.
1326

1327 - Distance Analysis
1328

1329 The minimum distances obtained were used to generate a histogram representing the distribution
1330 of observed distances. The distance histogram was divided into several bins (distance intervals).
1331 The first bin, representing the shortest distances (>0.5nm), was specifically analyzed to identify
1332 the closest contacts between proteins and PI4P. The contact distances calculated with gmx for
1333 each domain (C2B, C2C, and C2D) were obtained as previously described in the minimum
1334 distance section. We performed an ANOVA test to statistically evaluate these contacts, ensuring
1335 all conditions such as homogeneity of variances and normality were met.
1336

1337 • Quantification and Statistical Analysis
1338

1339 Statistical analyses were performed in GraphPad Prism (GraphPad Software). All experiments

1340 were independently repeated a minimum of 2-3 times (indicated in figure legends). Data are expressed as median unless specified in the figure legends.
1341 References

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