An Actin Remodeling Role for Arabidopsis Processing Bodies Revealed by their Proximity Interactome

Resource An actin remodeling role for Arabidopsis processing bodies revealed by their proximity interactome Chen Liu 1 , Andriani Mentzelopoulou 2,3 , Amna Muhammad 1,4 , Andriy Volkov 5 , Dolf Weijers 5 , Emilio Gutierrez-Beltran 6,7 & Panagiotis N Moschou 1,2,3,* Abstract Cellular condensates can comprise membrane-less ribonucleoprotein assemblies with liquid-like properties. These cellular condensates influence various biological outcomes, but their liquidity hampers their isolation and characterization. Here, we investigated the composition of the condensates known as processing bodies (PBs) in the model plant Arabidopsis thaliana through a proximitybiotinylation proteomics approach. Using in situ protein–protein interaction approaches, genetics and high-resolution dynamic imaging, we show that processing bodies comprise networks that interface with membranes. Surprisingly, the conserved component of PBs, DECAPPING PROTEIN 1(DCP1), can localize to unique plasma membrane subdomains including cell edges and vertices. We characterized these plasma membrane interfaces and discovered a developmental module that can control cell shape. This module is regulated by DCP1, independently from its role in decapping, and the actin-nucleating SCAR–WAVE complex, whereby the DCP1–SCAR–WAVE interaction confines and enhances actin nucleation. This study reveals an unexpected function for a conserved condensate at unique membrane interfaces. Keywords ARP2–ARP3; condensates; LLPS; plasma membrane domains SCAR–WAVE Subject Categories Cell Adhesion, Polarity & Cytoskeleton; Plant Biology; RNA Biology DOI 10.15252/embj.2022111885 | Received 15 June 2022 | Revised 10 January 2023 | Accepted 18 January 2023 | Published online 6February 2023 The EMBO Journal (2023)42:e111885 Introduction Proteins may participate in multivalent interactions with themselves or one another. Multivalency can depend on weak interactions between charged residues, dipoles, and/or aromatic groups often displayed by so-called “intrinsically disordered regions” (IDRs). These weak interactions can promote their dissociation from the bulk protein pool to form droplet-like assemblies through, for example, liquid–liquid phase separation (LLPS; Beutel et al,2019). LLPS is a state favoring condensation through weak intra- or intermolecular interactions. We use the term “condensates” hereafter to describe such proteinaceous assemblies. LLPS occurs when the bulk concentration exceeds a threshold above which molecules spontaneously partition into condensates that can resemble droplets due to surface tension. Transient or more stable condensates form in the nucleus, cytoplasm, or plasma membrane (PM) interfaces, for example, the animal-specific junction adherent molecules (Beutel et al,2019; Zaccara & Jaffrey, 2020). Condensates can coarsen, increase or decrease in size over time on membrane surfaces (Snead et al,2022). Likewise, the archetypal and evolutionarily conserved condensates known as processing bodies (PBs), which are rich in proteins and RNA and modulate RNA silencing, decapping and decay, undergo fission at membrane surfaces of the endoplasmic reticulum (ER) through an unknown mechanism (Lee et al,2020). In accordance with their functions, PBs contain decapping and exosome complexes, deadenylases, RNAs, and RNA-binding proteins. In PBs, including in plants, proteins such as the RNA decapping complex comprising mainly DECAPPING 1 (DCP1) and DCP2, and the proteins DCP5, VARICOSE (VCS), Protein Associated with Topoisomerase II 1 (PAT1), as well as the EXORIBONUCLEASE 4 (XRN4) localize there (Xu et al,2006; Xu & Chua, 2009; Rymarquis et al,2011; Roux et al,2015). Although systematic studies of plant PBs are lacking, animal PBs appear to be long-term storage sites for mRNAs poised to be released for translation following specific cues related to stress, metabolism, and translation capacity. Albeit readily visible in cells, as aforementioned, condensates such as PBs depend on a meshwork of weak interactions and lack a surrounding membrane, which makes their isolation challenging. Proximity-dependent biotin ligation (or PDL) harnesses covalent biotinylation of interacting proteins with or near neighbors of a 1Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden 2Department of Biology, University of Crete, Heraklion, Greece 3Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece 4University Institute of Biochemistry and Biotechnology, PMAS-Arid Agriculture University Rawalpindi, Rawalpindi, Pakistan 5Laboratory of Biochemistry, Wageningen University and Research, Wageningen, The Netherlands 6Instituto de Bioqu ımica Vegetal y Fotos ıntesis, Universidad de Sevilla and Consejo Superior de Investigaciones Cient ıficas, Seville, Spain 7Departamento de Bioqu ımica Vegetal y Biolog ıa Molecular, Facultad de Biolog ıa, Universidad de Sevilla, Sevilla, Spain *Corresponding author. Tel: +30 2810 394073; E-mail: [email protected]; panagiotis.m[email protected] Ó2023 The Authors. Published under the terms of the CC BY 4.0license The EMBO Journal 42:e111885 |2023 1of 26 Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. particular prey protein; we and others have recently established PDL approaches in various plants (reviewed in Zhang et al,2022). By bypassing the need to retain native interactions for their identification, PDL holds promise to delineate the organizational and functional principles of condensates. Inspired by PDL applications for the elucidation of condensates in non-plant models (Youn et al,2018), we established a PDL pipeline to determine the protein composition of condensates in plants, using PBs as a proof of concept. We show here that PBs are organized into both known and previously unknown functional modules. We further discovered that PBs components interface with PM domains, accumulating at cell edges or vertices. The suppressor of the cAMP receptor (SCAR)–WASP family verprolin homologous (WAVE) complex modulates this interface by recruiting DCP1. The SCAR–WAVE–DCP1 link enhances actin nucleation likely through the actin-related protein complex 2/3 (ARP2–ARP3; Kim et al,2021; Lee & Szymanski, 2021; Qin et al,2021). This SCAR–WAVE–DCP1 nexus functions as a coordinated cellular system that culminates in growth regulation. Results A bait for PB proteome capture To establish an approach for determining the protein composition of condensates in plants, we focused on PBs as a proof of concept, as non-plant PBs are omnipresent. A stepwise PDL-based approach might be the most appropriate for determining the composition of condensates, as we assumed that incorporating a standard affinity purification (AP) step before a subsequent PDL step might help distinguish between strong or direct versus the more specific interactions for condensates which are transient and weak. We thus used DCP1 as a bait tagged with both the sequence encoding for FLAG peptide and the biotin ligase “TurboID” as a construct encoding a chimeric DCP1-TurboID-6xHis-3xFLAG (driven by the 35S promoter; 35Spro:DCP1-TurboID-HF). The FLAG can be used for AP of DCP1 through a-FLAG beads and the TurboID for PDL. We describe the pipeline in Fig 1A and the technical details in the Materials and methods. As a specificity control for our assays, we used lines expressing GFP fused to 3xFLAG and TurboID (35Spro:GFP-TurboID-HF); the TurboID was described in (Arora et al,2020). DCP1-TurboID-HF was functional, showing higher expression levels (~25%, P=0.047, ANOVA) than were seen in wild-type (WT) seedlings, it could rescue the decreased size of adult plants of the dcp1-3 mutant (Martinez de Alba et al,2015), and could lead to efficient proteome biotinylation (Fig EV1A–C). Therefore, this DCP1 fusion is a physiologically relevant bait with which to explore the PB interactome. APEAL captured the PB proteome We asked whether the AP/PDL combination using the DCP1 bait could indeed capture both direct interactions but also weak ones. We define the PDL results hereafter as the PBs “proxitome.” After the AP step, we detected biotinylated proteins in the remaining supernatant (Figs 1A and EV2, corresponding to the “flow-through 1”). We thus decided to further consider proteins obtained from both the AP and PDL steps; this tandem analytical approach is referred to hereafter as “APEAL” (for tandemly coupled Affinity Purification with Proximity-dEpendent LigAtion steps). We coupled APEAL with nano-liquid chromatography–tandem mass spectrometry (nano-LC–MS/MS) in biological duplicates on DCP1 baits using 4-week-old rosettes grown under heat stress (HS) conditions (37°C for 2 h) and non-stress (NS) conditions. We selected HS treatment to benchmark APEAL because HS induces a size and number increase in PBs and likely a change in their composition (Gutierrez- Beltran et al,2015), and it enhances RNA decapping through the increased association of DCP1–DCP2 with polysomes (Merret et al,2013). We observed that HS does not significantly affect biotinylation levels of DCP1 (Fig EV1B, i.e., “24 h” vs. “HS”, the two samples obtained under the same setting: 24 h application of biotin with or without HS), suggesting that any differences in the PB proteome between NS and HS conditions in the APEAL may be of biological relevance. Proteome analyses yielded almost equivalent numbers of protein hits for AP and PDL steps (Fig EV3). Unexpectedly, and despite the size increase in PBs noted above, HS led to fewer proteins being captured in PDL/HS from DCP1, suggesting that upon HS the DCP1 molecules may be spatially further confined interacting will fewer proteins (see below for a possible explanation). To test for enrichments across the different samples, we applied the same criteria described in our previous work to define the high-confidence proximity interactions (Arora et al,2020). Briefly, we considered only interactions present in both biological replicates (log 2 [fold change] ≥1, n=2, false-discovery rate [FDR] =0.05; Source Files 1 and 2, for unfiltered and filtered data, respectively). To compare the AP and PDL datasets, we also assigned LFQ (label-free quantitation) and iBAQ values (intensity-based absolute quantification, Source Files 3 and 4; Cox et al,2011) to the semi-quantitative peptide enrichments and reiterated interactions. The LFQ/iBAQ assignments produced similar results to the semiquantitative analysis. An inspection of protein hits in the PDL step confirmed an enrichment for conserved PB core components (Fig 1B; log 2 FC ˃1; Gutierrez-Beltran et al,2021). As a cautionary note, we do not define the PB core as a structural entity with topological essence (e.g., with the PB center formed as an immiscible liquid), but rather as an indispensable set of critical or accessory proteins that comprises part of the decapping machinery. Intriguingly, the AP step failed to enrich for PB core components, suggesting that many weak interactions are not retained, thus validating our dual approach (Fig 1B; “AP” vs. “PDL”). Importantly, APEAL showed no enrichment for proteins exclusively associated with the other condensate known as stress granules (SGs; e.g., RNA-BINDING PROTEIN 47B [RBP47]), despite the putative physical links between the two condensates during HS (Gutierrez-Beltran et al,2021). One exception was the class of RNA helicases which are present in both SGs and PBs (Fig 1B; log 2 FC ≥1.5, RH6, RH8, RH12, and the newly discovered component of PBs RH52 herein). The absence of other SG components confirmed that APEAL, at least, in this case, can provide the required spatial resolution to specifically resolve the composition of PBs and highlights the functional differences between the PBs and SGs. Network analyses reveal that PBs interface with membranes We integrated our PDL hits with delimited direct protein–protein interaction (PPI) data from the STRING (Search Tool for the 2of 26 The EMBO Journal 42:e111885 |2023 Ó2023 The Authors The EMBO Journal Chen Liu et al Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. Retrieval of Interacting Genes/Proteins) database (Jensen et al,2009), revealing that the PDL dataset forms a denser PPI network than that obtained with AP. The average numbers of interactions from PDL per protein were 5.4 (P=5.6e 11 ) and 4.1 (P=2.1e 10 ) for NS and HS, respectively (Fig 1C). The PDL dataset also showed a different profile in terms of interactions from AP, while different interactions were observed when HS and NS were compared (Figs 1D and 2A). Consistent with the expected incremental formation of PBs during HS, the term “PΒs” was more enriched in the PDL dataset upon HS when compared to NS (P=6.9e 2 vs. 6.9e 22 ). We validated the results of the PDL step using bi-fluorescence complementation (BiFCs; concept described in Fig EV4A, negative control XRN3, P=0.19 to <5.7e 4 ) and colocalization analyses in the heterologous Nicotiana benthamiana transient expression system, showing the successful identification of novel components of PBs or DCP1 interacting proteins (Fig EV4A and B; nine out of nine proteins tested). We detected a partial overlap between our datasets and other published proteomics datasets that analyzed PM composition by AP with various baits (Source Files 5 and 6; overlap 27.1%; Fernandez-Calvino et al,2011; Bernfur et al,2013; Rutter & Innes, 2017), and with endomembranes (overlap 9–32.8%; Heard et al,2015), suggesting that PBs may interface Figure 1. The pipeline of the APEAL approach. A Overview of the APEAL pipeline. Upon 24 h biotin feeding and treatment (supplied with 50 lM biotin directly into leaves of 4-week-old plants by syringe infiltration), total proteins are extracted from infiltrated leaves. The proteome is subjected to AP-immunocapture of the FLAG tag. In the AP step, some of the captured proteins will be biotinylated. The PDL step uses the leftover supernatant from the AP step and captures biotinylated proteins with streptavidin beads. PPI, protein–protein interactions; AP, affinity purification; PDL, proximity-dependent biotin ligation; FLAG IP, FLAG-beads immunoprecipitation; DYNA-IP, streptavidin-bead immunoprecipitation. We use the term “proxitome,”to describe the proteins captured by the PDL step of APEAL. These proteins may not physically interact with DCP1. B Heatmap showing the “core processing body (PB)”(magenta) components identified and other linked proteins. RBP47 is a stress granule marker (SGs; blue). RH52 is a new helicase identified as a PB component (green arrow). The scale on the right shows log 2 FC of protein abundance. Note that only the PB core components were enriched in the PDL (i.e., the proxitome, log 2 FC ~1or above) but not in AP. Furthermore, heat stress (HS) increased the enrichment of some PB core components. C Comparison of AP/PDL interacting networks produced from APEAL. STRING density plots of pairwise interactions between proteins obtained from the AP or PDL steps (combined interactions found in non-stress [NS]/[HS]). Note that PDL produces an overall denser interaction network (under standard parameters, the same number of proteins was selected for AP/PDL). D Venn diagram showing the proteins identified for PDL and AP in NS and HS samples (PPIs fulfilling the criterion log 2 FC >1). Source data are available online for this figure. Ó2023 The Authors The EMBO Journal 42:e111885 |2023 3of 26 Chen Liu et al The EMBO Journal Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. with membranes. In comparison with a recent study examining DCP1 and DCP2 interactions through AP, we found an overlap of up to 36.3% (Schiaffini et al,2021). Notably, this comparison showed a lower-than-expected overlap with our datasets, while in this previous study similar membrane-related proteins were missing. Although in this previous study, the stabilizer of weak interactions formaldehyde was used, because it has the shortest span (~2–3  A) of any known crosslinker (Nadeau & Carlson, 2007), it may not be the best option to stabilize interactions at the interface between condensates and membrane proteins. More crosslinkers with various spans will need to be tested, but we speculate for now that proxitomes may expand identifications of new proteins in PPI studies. Furthermore, our datasets suggested that PB may interface with membranes; we clarify this in detail below. We tested hierarchical linkages between the networks we generated by integrating the data from STRING with our datasets using hypergeometric tests (FDR =0.05). We assigned four dense and interconnected subnetworks to PBs according to overrepresentations in AP and PDL datasets from both HS and NS conditions (Figs 2and EV5; note that in volcano plots in PDL the DCP1 was not the most abundant). Subnetwork 1 is related to RNA metabolism (Fig 2B). In subnetwork 2, we obtained hits for phytohormone signaling, defense attenuation, translation, and metabolism (Fig 2C). Subnetwork 3 comprises an unexpected network for PBs, with hits linked to membrane remodeling and trafficking (Fig 2D); finally, subnetwork 4 includes another surprising network of proteins linked to the cytoskeleton and in particular actin remodeling (Fig 2E and Source File 7). In the Appendix text, we succinctly Figure 2. APEAL captured the PB proteome and proxitome. A Volcano plots showing significantly enriched proteins in NS and HS conditions from the AP and PDL APEAL steps. Selected proteins are indicated in magenta and are encoded by genes that belong to the identified subnetworks described in (C) and (D). Magenta indicates enrichment in HS samples; cyan indicates depletion in HS samples. B–E Gene Ontology (GO) enrichment analyses of the APEAL results, divided into four subnetworks. Note the terms related to vesicle trafficking and actin remodeling. A more detailed description is provided in Fig EV5and in the Appendix text. Note that signal transduction proteins, metabolism-related proteins, and vesicle trafficking proteins evade PBs during HS (Fig EV5, reduced hit number), while the opposite pattern is observed for actin and RNA metabolism subnetworks. FDR, false discovery rate. Important links described below are indicated (i.e., to the SCAR–WAVE/ARP2–ARP3(and the component NAP1which is part of the SCAR–WAVE and links to autophagy), cytoskeleton, PB core, and cell-wall-related metabolism). Source data are available online for this figure. 4of 26 The EMBO Journal 42:e111885 |2023 Ó2023 The Authors The EMBO Journal Chen Liu et al Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. describe these networks, from which testable hypotheses may arise for future studies. DCP1interfaces with plasma membrane domains at the cellular face independently of its role in decapping The interfacing between condensates and membranes can be functionally important by regulating protein activities, as has been shown for animal cells (Snead et al,2022). We thus asked whether the links between PBs and subnetwork 3, which suggested membrane functions, might relate to the decapping function of PBs. To observe membranes in detail and expedite analyses, we focused on the PM and used total internal reflection microscopy (TIRF-M), which is suitable for viewing cell surface processes due to shallow illumination penetration (decay constant ~100 nm). TIRFM allowed us to observe occasional stalling and fusion of DCP1-GFP-positive droplets with one another at the lateral PM interface of meristematic root epidermal cells (Fig 3A and B). Though encouraging, we note here that these results should not be interpreted as a direct association between DCP1 and membrane lipids. As a cautionary note also, the lateral PM used for imaging due to the TIRF-M inherent limitations which do not allow observation of apicobasal membranes, may not reflect the exact situation of these membranes. Furthermore, due to the swallow illumination depth, TIRFM does not allow observation of cells below the epidermis or the root cap in roots (e.g., cortex). Mean squared displacement (MSD) analyses, which track particles in two dimensions, showed that DCP1-positive droplets become more stable with time, after an initial Brownianmotion diffusion at the PM (Fig 3B, inset; track analyses). This tracking also showed that stable DCP1-positive droplets lose signal with time (Fig 3B, kymograph). This result suggested that DCP1- positive droplets may diffuse to form 2D domains at the inner face of the PM and highlights that subnetwork 3 may be of biological relevance. High-speed super-resolution confocal microscopy (~120 nm axial resolution and 40 frames per second) and fluorescence partition analyses of the obtained micrographs between the PM and the overall signal at the cytoplasm (in PBs or the dilute phase) detected a fraction of mScarlet-DCP1 at the PM (marked here with the auxin efflux carrier PINFORMED2 [PIN2]), regardless of the promoter driving the DCP1 (Fig 3C, the constitutive in meristems RPS5a, the strong 35Spro or the native DCP1pro; both N- or C-tagged lines). Other core PB components were associated with the PM either very transiently (in dividing cells) or not at all (Appendix Fig S1A). We further observed that DCP1 can exhibit a polar behavior localizing at apical or basal domains of the PM, which was not observed for other core PB components (Fig 3C, lower right for polarity, and Appendix Fig S1). Moreover, the addition of cycloheximide (CHX), known to dissolve PBs as they are in dynamic equilibrium with polysomes (Gutierrez-Beltran et al,2015), decreased PB numbers and led to greater DCP1 localization at the PM (Appendix Fig S1B). By generating and using DCP1 tagged with the photoconvertible molecule EosFP to track in vivo DCP1 molecules (Wiedenmann et al,2004;UBQ10pro:EosFP-DCP1; photoactivatable green-to-red; Appendix Fig S1C caption for method details), we confirmed CHX results suggesting that DCP1 can shuttle between PBs and the PM (Appendix Fig S1C, and edges as described below). We cannot though discount the possibility that a fraction of DCP1 is targeted to the PM independently from PBs. Taken together, these results indicate that DCP1 localization at the PM is in a dynamic equilibrium with PBs. Notably, some cells accumulated more overall DCP1-GFP at the PM than others, while in some cases we observed a spatial restriction of the signal at cell edges not seen for other PBs proteins or PIN2 (e.g., Fig 3C and Appendix Fig S1B). We use the term “edge” in the geometric sense of an intersection between two faces of a polyhedron, rather than of a periphery or front (Kirchhelle et al,2016). This variability of localization prompted us to examine in detail in which cells DCP1 can be found on membranes, including the edges. To expedite the analyses here, we divided the roots into three regions (see also Fig 3A). Dynamic 3D imaging of DCP1-GFP in these regions showed that the probability to find DCP1 at cell edges was higher far from the quiescent center (QC) along the proximodistal root axis (as defined in Fig 3A), where DCP1 can be further confined at the inner face of the PM at vertices (Fig 4A, e.g., in region 3). Like “edge”, the term “vertex” here is used in the geometric sense to define the internal angular point of a polygon and is defined as an intersection between the three cell edges. Under HS conditions, DCP1-GFP abundance decreased at the PM at the expense of PBs but increased along the cell edges (Fig 4B). Our results suggested a dynamic competition between PBs and vertices exclusively for the DCP1 component. As DCP1 localization at the edge or vertex was unaffected by the presence of CHX (Appendix Fig S1B, arrowhead), this result implied that DCP1 did not form decapping complexes at PM interfaces. To further validate this result, we used ultra-fast live-cell imaging super-resolution microscopy showing that DCP1–decorated PBs undergo fission at the PM plane, probably to remodel PBs and release DCP1 (Fig 4C). This result is consistent with the diffusible loss of material from PBs associated with the PM observed by TIRF- M (Fig 3B; at the same regions, 2 and 3) and likely implied that DCP1 dissociates from DCP2, although whether this dissociation is a prerequisite for the further loss of material requires further studies. Likewise, in animals, PBs undergo fission by the membranous ER sheets (Lee et al,2020), but it is also unclear whether ER plays a role in splitting PBs at the PM. Accordingly, when we tested the dynamic interaction between DCP1 and DCP2, by sensitized emission Fo ¨rster resonance energy transfer (SE–FRET), we did not detect DCP1–DCP2 interaction at the PM, vertex, or edge (Appendix Fig S2A). However, DCP1 did show a weak and transient association with the PBs core protein PAT1 at the PM (but not at the edge or vertex; Appendix Fig S2A). As the decapping complex comprises DCP1 and DCP2 as minimal components (Charenton et al,2016), together with the CHX data, these results argued against the formation of a decapping complex at the PM or on edges/vertices. To validate the lack of association between DCP1 and DCP2 at membranes, as FRET is sensitive to stoichiometry and may also fail to capture very transient interactions, we exploited the power of quantitative 3D proximity ligation assays (PLAs; Teale et al,2021; preprint: Liu et al,2022). PLA uses complementary oligonucleotides fused to specific antibodies to determine the frequency with which proteins of interest find themselves nearby (<40 nm). When the proteins of interest interact or are nearby, PLA leads to a spot-like signal (Appendix Fig S2B, left). In the PLA assay, DCP1 and DCP2 interacted or were nearby only in the Ó2023 The Authors The EMBO Journal 42:e111885 |2023 5of 26 Chen Liu et al The EMBO Journal Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. Figure 3. DCP1protein interfaces with the plasma membrane. A Diagram of a root showing the three developmental regions (1–3) under examination in this study: stem cell niche (SCN; region 1), meristematic zone (MZ; region 2), and transition zone (TZ)-differentiation zone (DFZ; region 3). The different cell types are color-coded. The region used in TIRF-M experiments is highlighted by the dashed magenta rectangle (region 3, see also B). B Representative TIRF-M of DCP1-GFP (DCP1pro:DCP1-GFP transgene) in the lateral PM of epidermal cells showing the transient attachment of DCP1-positive puncta to the PM. Yellow arrowheads denote a PB that shows motility at the PM focal plane; blue arrowheads show a PB that transiently localizes to the PM. Scale bars: 2lm. The corresponding kymographs are shown to the right. Right: distribution of immobile and mobile DCP1molecules relative to the motility log(D) value of 0.75 (threshold; see Materials and Methods for details), in NS or HS conditions (D, diffusion coefficient). Inset: individual trajectories of mobile DCP1-GFP in NS and HS conditions (500 frames, n=120), showing a combination of directional and Brownian motion for both NS/HS. The green arrowheads denote the beginning of the NS and HS tracks for DCP1-GFP. C Representative confocal micrographs from lines co-expressing RPS5apro:HF-mScarlet-DCP1and PIN2pro:PIN2-GFP (epidermal cells, region 3, Scale bars: 5lm). Bottom: polarity index of DCP1in root meristematic cells (compared to propidium iodide (PI) and tubulin staining of root cells; N,biological replicates =3roots, n=13 cells). Polarity index is calculated as the ratio of average of apical and basal VS lateral side of fluorescence signal intensity of the root epidemies cells. The arrowhead in PIN2indicates the cell plate or PM in DCP1. Right: representative confocal micrographs showing that PM localization is independent of the promoter used (DCP1pro, 35Spro; region 2, epidermal cells, or RPS5apro on the lower right). The details from the inset show increased localization at the cell edge (discussed later). mSc, mScarlet. Scale bars: 7lm. Data information: In C, Pvalues were determined by Wilcoxon. Upper and lower lines in the violin plots when visible, represent the first and third quantiles, respectively, horizontal lines mark the median and whiskers mark the highest and lowest values. Source data are available online for this figure. 6of 26 The EMBO Journal 42:e111885 |2023 Ó2023 The Authors The EMBO Journal Chen Liu et al Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. cytoplasm but not at the PM (Fig 5). We also tested DCP1 against PAT1 (positive control) and the auxin efflux carrier PINFORMED7 (PIN7; negative control) to assess the reliability of PLA in Arabidopsis roots, with the prediction that fewer interactions should occur when target protein pairs have increasingly discrete accumulation domains (i.e., PIN7; absent in APEAL, Appendix Fig S2B and C). Unexpectedly, through this approach, we established that DCP1 and DCP2 form complexes also in the nucleus (Fig 5, inset 2), as was shown in yeast (Saccharomyces cerevisiae), where they act as a decapping reservoir (Tishinov & Spang, 2021). Together, PLA and SE–FRET analyses speak against the existence of a localized decapping activity at the PM. Furthermore, our analyses show that PLA assays can help in validating proxitomes. Figure 4. DCP1protein accumulates at edges and then vertices during development. A Left: Gradual edge or vertex accumulation of DCP1-GFP in three different root regions. DCP1signal intensity among the three different regions, at the edge/vertex (epidermis). Right: z cross-sectional images of DCP1-GFP (green) in the whole root compared to FM4-64 staining (magenta, staining membranes). The circular plots indicate the average DCP1localization (regions 1–3;N,biological replicates =3,n=10 cells; comparing regions 1–2and 2–3). The 3D-rendered images (PIN2-GFP vs. mSCarlet-DCP1) show the localization of mScarlet-DCP1at edges/vertices in two different regions (in comparison to PIN2-GFP signal which decorated almost evenly the PM). Scale bars: 20 lm. Arrowhead denotes the edge (region 2) and the vertex (region 3) decorated by mScarlet-DCP1(also in the z cross-sectional image). Note in a single cell file, how the localization from the PM changes to the edge, as indicated, along the proximodistal axis. B Representative confocal micrograph of DCP1-GFP (DCP1pro:DCP1-GFP, transgene) in root meristematic cells under NS/HS conditions (region 2). Scale bars, 5lm. Note the depletion of DCP1from the PM upon HS, but the increased edge/vertex signal (yellow arrowheads denote the vertex signal). Right: DCP1signal intensity at the PM or edge/vertex (N,biological replicates =3,n(pooled data of 3biological replicates)=18–23 PMs or edges/vertices). C Representative confocal images showing fusion (coarsening), fission, and growth of PBs (DCP1-positive) at the PM (region 3). Right: states of PBs (dynamic: fusion and fission and non-dynamic: stable; N,biological replicates =2,n(pooled data of 3biological replicates)=6–8PBs). As a cautionary note, the “stable”PBs may not show dynamicity in the imaging time used (~3–5min) but later, may do. Data information: In (A), *P<0.05 was determined by a nested t-test. In (B), Pvalues were determined by the Kolmogorov–Smirnoff, while in (C) by one-way ANOVA. Upper and lower lines in the violin plots when visible, represent the first and third quantiles, respectively, horizontal lines mark the median and whiskers mark the highest and lowest values. Source data are available online for this figure. Ó2023 The Authors The EMBO Journal 42:e111885 |2023 7of 26 Chen Liu et al The EMBO Journal Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. SCAR–WAVE recruits DCP1at the cell edge/vertex Given the reversible association of DCP1 with the PM (e.g., in HS), we postulated that DCP1 is not inserted there. We first tested whether DCP1 accumulates at the PM, edges, or vertices, through the secretory pathway by incubating seedlings with brefeldin A (BFA, 50 lM), a fungal toxin that prevents vesicle formation for exocytosis by inhibiting ADP ribosylation factor GEFs (ARF-GEFs; Satiat-Jeunemaitre et al,1996). DCP1-GFP did not accumulate intracellularly upon BFA treatment (Appendix Fig S3), and it lacks a signal peptide or a transmembrane domain, suggesting that it is not inserted in the PM and likely is not a secreted protein. The accumulation of DCP1 during development at cell edges and vertices prompted us to examine the underlying mechanism by which DCP1 is recruited there. We postulated that proteins with which DCP1 interacts could regulate this localization. To this end, we compared DCP1 localization to that of other proteins that localize at cell edges/vertices and are enriched in the APEAL (i.e., SOSEKI3 [SOK3], which regulates a cellular coordinate geometric system (van Dop et al,2020) with log 2 FC =0.58 for NS and 2.0 for HS; and the SCAR–WAVE complex (Dyachok et al,2008) that regulates actin nucleation). In addition, as a negative control, we used the edge-localizing Ras-related protein Rab-A5c (Kirchhelle et al,2019), which we did not identify by APEAL. SOK3 localized at the cell division zone (the cell plate fusion site), unlike DCP1, which was absent from this site (Appendix Fig S4A and B, region 1). Later in development, SOK3 showed some signal collinearity with DCP1 at edges/vertices (Pearson’s correlation coefficient =0.87, region 3). In contrast, Rab-A5c showed little colocalization with DCP1 at all stages examined (Appendix Fig S4C). We further observed that neither the loss of SOK3 function (from a sok3 mutant), the simultaneous loss of SOK1/SOK3 functions (from genome editing in the sok3 mutant; details in Materials and methods), nor Rab-A5c depletion (through a dominant-negative dexamethasone-inducible expression of inactive Rab-A5c; Kirchhelle et al,2016) resulted in changes in DCP1 localization at the PM or the edge/vertex (Appendix Fig S4B and C, in live imaging or through detection by a- DCP1; details for the antibody in Materials and methods). These data suggested that neither SOK3 nor Rab-A5c recruit DCP1 at vertices or edges. In sharp contrast with Rab-A5c or SOK3, we observed almost perfect signal collinearity at vertices/edges for mCherry and GFP signals in cells co-expressing SCAR2-mCherry (with log 2 FC =2.32 for NS and 3.62 for HS, respectively) and DCP1-GFP or BRK1-YFP (BRICK1; components of the SCAR–WAVE complex; Dyachok et al,2008) with mScarlet-DCP1 (Fig 6A). The SCAR2 signal though at the cell plate was weak and we were unable to draw conclusions about colocalizations there. Of note, other SCAR–WAVE components like PIR121/SRA1, NAP1, and GRL/NAP125 were also highly enriched in PDL datasets, especially upon HS (log 2 FC ≥2 for NS and 3.5 for HS; Fig EV5 and Source Data 7). Accordingly, upon HS, as DCP1 association with the PM decreased, we observed a stronger colocalization at edges/vertices between SCAR2 and DCP1 (Fig 6A). This result explains well the increase in DCP1 signal at edges/vertices in HS and the APEAL results suggesting a stronger association of SCAR–WAVE/DCP1 under HS. Figure 5. DCP1interacts with DCP2in a PLA assay occasionally in the cytoplasm but not at the PM. Confocal micrographs showing single optical sections PLA-assays producing signal that resembles spots. The antibodies used were anti-FLAG/anti-GFP detecting the HF- mScarlet-DCP1/ DCP2-YFP, respectively (RPS5apro:HF-mScarlet-DCP1and 35Spro:DCP2-YFP). Inset 1: magnification showing the colocalization of PLA spots with DCP1or DCP2signals (colocalization and Pearson’s correlation coefficient PCC value for the spots shown). The dotted white line in the PLA channel corresponds to the PM plane. Inset 2: positive PLA signal for DCP1and DCP2in the nucleus. “n1-n3”correspond to nuclei regions (green circles). On the right, note the PLA spot nearby the nucleus (“detail of PLA signal”). The chart shows the quantification of PLA spots per cell at puncta (cytoplasm) or on the PM (N,biological replicates =3,n(pooled data of 3biolog- ical replicates)=16–33 cells). As a cautionary technical note, the cytoplasmic, nuclear or PM “spots”do not connote physiologically relevant puncta, condensates, or PM clusters. Scale bar: 20 lm/10 lm for the insets. Data information: In (A), *P<0.05 was determined by one-way ANOVA. Upper and lower lines in the violin plots when visible, represent the first and third quantiles, respectively, horizontal lines mark the median and whiskers mark the highest and lowest values. 8of 26 The EMBO Journal 42:e111885 |2023 Ó2023 The Authors The EMBO Journal Chen Liu et al Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. We also investigated whether the colocalization between DCP1 and SCAR–WAVE, reflected an interaction. Indeed, DCP1 and SCAR2, or DCP1 and BRK1, interacted in FRET assays in edges/ vertices but not in PBs (Fig 6B, region 3). To ascertain these interactions, we used the quantitative 3D PLA assay (Fig 6C). PLA determined that DCP1 and SCAR–WAVE components mainly interact at Figure 6. DCP1cooperates with the SCAR–WAVE complex. A DCP1colocalizes with the SCAR–WAVE components SCAR2and BRK1. Representative confocal micrographs showing colocalization between DCP1and SCAR2or DCP1 and BRK1(in lines coexpressing DCP1pro:DCP1-GFP and SCAR2pro:SCAR2-mCherry or RPS5apro:HF-mScarlet-DCP1and BRK1pro:BRK1-YFP; arrowheads indicate colocalization at cell edges or vertices) in root epidermal cells (regions 2–3). Right: relative signal intensity profiles of DCP1or SCAR2at vertices. Colocalization at these regions was also calculated (PCC; N,biological replicates =2,n=4–10 edges/vertices in regions 2–3; a slight increase was observed in HS, 0.78 in NS vs. 0.87 in HS). Scale bars: 10 lm. B Representative confocal micrographs with acceptor photobleaching-FRET efficiency between SCAR2-mCherry (up) or BRK1-mRuby (down) and DCP1-GFP (epidermal cells, regions 1and 3). Right: normalized FRET efficiency between SCAR2or BRK1and DCP1, respectively, among the different developmental root regions (N,biological replicates =2,n=16 cells). Scale bars: 3lm. C Representative confocal micrographs showing PLA spots produced by a-GFP/a-RFP in DCP1-GFP/SCAR2-mCherry lines and DCP1-GFP/BRK1-mRuby lines, a-FLAG/a-GFP in HF-mScarlet-DCP1/SPI-YPet (SPIRRIG [SPI] is a negative control as it localizes to PBs only during salt stress and was not found in the APEAL). In the SPI PLA, a high contrast inset is presented. Right: number of PLA spots per cell (N,biological replicates =3,n(pooled data of 3biological replicates)=14–33 cells). In the merged images, the cell contours are shown (light green transparent). Scale bars: 5lm. D Representative confocal micrographs showing DCP1localization detected by a-DCP1in the wild type (WT) or the scar1scar2scar3scar4(scar1234) quadruple mutant or in live-cell imaging of mScarlet-DCP1(RPS5apro:HF-mScarlet-DCP1)inWTorbrk1mutant (bottom right; epidermal cells, region 3for a-DCP1and 2for live-cell imaging). The arrowheads denote the lack of robust DCP1localization in scar1234 at the edge/vertex. Small panels (insets) at right show details corresponding to the regions delineated by dashed lines, where arrowhead denotes the edge signal of DCP1in WT; scale bars, 1lm. Bottom: percentage of cells with proper edge/vertex localization and quantification of PB numbers (DCP1-foci; N,biological replicates =3,n(pooled data of 3biological replicates)=18–35 cells). Scale bars, 5lm. Data information: In (B), Pvalues were determined by Kruskal–Wallis, and the comparisons are among the scar1234 to the corresponding WT samples. In (C and D), P values were determined by Wilcoxon. PCCs are means s.d. Upper and lower lines in the violin plots when visible, represent the first and third quantiles, respectively, horizontal lines mark the median and whiskers mark the highest and lowest values. Source data are available online for this figure. Ó2023 The Authors The EMBO Journal 42:e111885 |2023 9of 26 Chen Liu et al The EMBO Journal Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. Reagents and Tools table (continued) Reagent/Resource Reference or Source Identifier or Catalog Number Arabidopsis thaliana Columbia arpc5SALK_123936.41.55 (Pratap Sahi et al,2017) Arabidopsis thaliana scar1234 Dyachok et al (2008), Chin et al (2021) Arabidopsis thaliana Columbia brk1Dyachok et al (2008), Chin et al (2021) Arabidopsis thaliana Columbia sok1sok3CRISPR double mutant/this paper Arabidopsis thaliana Columbia SCAR2pro:SCAR2-mCherry Chin et al (2021) Arabidopsis thaliana Columbia BRK1pro:BRK1-mRuby3Chin et al,2021 Arabidopsis thaliana Columbia BRK1pro:BRK1-YFP Chin et al,2021 Arabidopsis thaliana Columbia UBQ10pro:Lifeact-mRuby Chin et al,2021 Arabidopsis thaliana Columbia UBQ10pro:Lifeact-mCherry Chin et al (2021) Arabidopsis thaliana Columbia PAT1pro:PAT1-GFP Roux et al (2015) Arabidopsis thaliana Columbia SOK3pro:SOK3-YFP van Dop et al (2020) Arabidopsis thaliana Columbia 35Spro:DCP2-YFP Jang et al (2019) Arabidopsis thaliana Columbia VCSpro:VCS-GFP Roux et al (2015) Arabidopsis thaliana Columbia UBQ10pro:DCP5-GFP Chicois et al (2018) Arabidopsis thaliana Columbia DEX˃RAB-A2c DN Kirchhelle et al (2016) Arabidopsis thaliana Columbia RAB-A5cpro:RAB-A5c Kirchhelle et al (2016) Arabidopsis thaliana Columbia UBQ10pro:EosFP-DCP1This paper Arabidopsis thaliana Columbia PIN2pro:PIN2-GFP Xu & Scheres (2005) Arabidopsis thaliana Columbia PIN7pro:PIN7-GFP Zhou et al (2010) Recombinant DNA pICSL86900-OD Addgene 86178 pICSL13002 Addgene 50266 pICH47751 Addgene 48002 pICH41414 Addgene 50337 pGWB560 Nakagawa et al (2007) pG5(RPS5apro:HF-GW-tagRFP) This paper pIF1(RPS5apro:HF-mScarlet-GW) This paper pIF22 (RPS5apro:mNeon-GW) This paper Codon-optimized TurboID Arora et al (2020) 35Spro:sGFP-TurboID-HF/WT Arora et al (2020) 35Spro:DCP1-TurboID-HF/WT This paper 35Spro:GFP-DCP1/WT Gutierrez-Beltran et al (2015) RPS5apro:ARPC5-tagRFP This paper RPS5apro:mNeon-ARPC5This paper pUbiCAS9-Red Durr et al (2018) pSITE17 (nYFP-GW) Chakrabarty et al (2007) pSITE18 (cYFP-GW) Chakrabarty et al (2007) pSITE18 (nYFP-GW)-DCP1This paper pSITE17 (cYFP-GW)-AT1G33050 This paper pSITE17 (cYFP-GW)-AT2G26920 This paper pSITE17 (cYFP-GW)-RH12 This paper pSITE17 (cYFP-GW)-FLXL1This paper 16 of 26 The EMBO Journal 42:e111885 |2023 Ó2023 The Authors The EMBO Journal Chen Liu et al Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. Reagents and Tools table (continued) Reagent/Resource Reference or Source Identifier or Catalog Number pSITE17 (cYFP-GW)-XRN3This paper pG5-ECT6This paper pG5-ECT4This paper pG5-VAP27-1This paper pG5-AT2G26920 This paper pG5-AT1G33050 This paper pG5-AT5G53330 This paper pG5-FLXL1This paper pG5-MLN51 This paper pG5-EIN2This paper Antibodies Mouse a-DCP1This paper Polyclonal mouse a-PAT1Roux et al (2015) Monoclonal a-FLAG® M2-Peroxidase (HRP) Sigma-Aldrich A8592 Monoclonal mouse a-FLAG® M2Sigma-Aldrich F1804 Polyclonal Rabbit a-Green Fluorescent Protein (GFP) Millipore AB10145 a-streptavidin-HRP Sigma-Aldrich GERPN1231 Monoclonal Mouse a-streptavidin Sigma-Aldrich 189730 Monoclonal Mouse a-GFP antibody Sigma-Aldrich SAB2702197 Monoclonal Mouse a-Red Fluorescent Protein (RFP) (clone RF5R) Agrisera AS15 3028 Monoclonal Rat a-tubulin (YL1/2) Santa Cruz Biotechnology sc-53029 Alexa Fluor® 488 phalloidin ThermoFisher Scientific A12379 Rhodamine Red TM -X (RRX) 570 AffiniPure Donkey a-Mouse IgG (H +L) Jackson ImmunoResearch 715-295-151 Rhodamine (TRITC) AffiniPure Donkey a-Rat IgG (H+L) Jackson ImmunoResearch 712-025-153 IRDye® 680LT Goat a-Mouse IgG (H +L) LI-COR 925-68020 IRDye® 800CW Goat a-Rat IgG (H +L) LI-COR 925-32219 Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey) Amersham NA934 Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep) Amersham NA931 Polyclonal rabbit a-His Oligonucleotides and sequence-based reagents Primers used for cloning This study Appendix Table S1 Primers used for verification of T-DNA mutants This study Appendix Table S2 Primers used for generating CRISPR mutants This study Appendix Table S3 Chemicals, enzymes, and other reagents a-FLAG® M2Magnetic Beads Sigma-Aldrich M8823 Dynabeads® M-280 Streptavidin Thermo Fisher Scientific 11205D PD-10 Desalting Columns Cytiva 17085101 Glutathione Sepharose 4B Cytiva 17075601 IgG sepharose Cytiva 170969-01 Ni-NTA Agarose Qiagen Ab 30210 HisPur TM Cobalt Resin ThermoFisher Scientific 89964 Duolink® In Situ PLA® Probe a-Rabbit PLUS Affinity purified Donkey anti-Rabbit IgG (H +L) Sigma-Aldrich DUO92002-100RXN Duolink® In Situ PLA® Probe a-Mouse MINUS Affinity purified Donkey anti-Mouse IgG (H +L) Sigma-Aldrich DUO92004-100RXN Ó2023 The Authors The EMBO Journal 42:e111885 |2023 17 of 26 Chen Liu et al The EMBO Journal Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. Reagents and Tools table (continued) Reagent/Resource Reference or Source Identifier or Catalog Number Duolink® In Situ PLA® Probe a-Goat PLUS Affinity purified Donkey anti-Goat IgG (H +L) Sigma-Aldrich DUO92003-100RXN Duolink® In Situ Detection Reagents Red Sigma-Aldrich DUO92008-100RXN Duolink® In Situ Detection Reagents FarRed Sigma-Aldrich DUO92013-100RXN Duolink® In Situ Probemaker PLUS Sigma-Aldrich DUO92009-1KT Duolink® In Situ Wash Buffers, Fluorescence Sigma-Aldrich DUO82049 Gentamycin sulfate Saveen Werner AB (Duchefa) G0124.0005 Ampicillin Saveen Werner AB (Duchefa) A0104.0010 Spectinomycin Saveen Werner AB (Duchefa) S0188.0005 Rifampicin Saveen Werner AB (Duchefa) R0146.0005 Kanamycin Saveen Werner AB (Duchefa) K0126.0005 Biotin Sigma-Aldrich B4501 Amiprofos methyl (APM) Sigma-Aldrich 3992 Latrunculin B (Lat B) Sigma-Aldrich L5288 Cytochalasin D (Cyt D) Santa Cruz Biotechnology sc-201442 Dithiothreitol (DTT) ThermoFisher Scientific R0861 Protease inhibitors cocktail Sigma-Aldrich P9599 PhosSTOP Roche 4906845001 Propidium Iodide Sigma-Aldrich P4170 IGEPAL CA-630 Sigma-Aldrich I8896 Isopropyl-b-D-1thiogalactopyranoside (IPTG) Sigma-Aldrich I6758 Imidazole Sigma-Aldrich 56748 TRIzol® Reagent ThermoFisher Scientific 15596018 30% Acrylamide/Bis Solution, 29:1Biorad 161-0156 4×Laemmli Sample Buffer Biorad 1610747 RiboLock Rnase Inhibitor ThermoFisher Scientific EO0381 DNase I, RNase-free ThermoFisher Scientific EN0521 Mesh SefarNitex 03-25/19 VectaShield Vector Laboratories H-1200 FM4-64 ThermoFisher Scientific T13320 Cycloheximide (CHX) Sigma-Aldrich C7698 Isoxaben Sigma-Aldrich 36138 DynaMag TM -2Magnet ThermoFisher Scientific 112321D Phenol: chloroform: iso-amyl alcohol (25:24:1) VWR 136112-00-0 Acetosyringone Sigma-Aldrich D134406 PageRuler TM Plus Prestained Protein Ladder, 10 to 250 kDa ThermoFisher Scientific 26619 Eppendorf 1.5ml Protein LoBind Microcentrifuge tubes VWR 525-0133 Bovine serum albumin (BSA) Sigma-Aldrich A7030 Deionized Formamide Sigma-Aldrich S4117 m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) ThermoFisher Scientific 22311 2-fluoro-N-[2-(2-methyl-1H-indol-3-yl)ethyl]-benzamide (CK-666) Sigma-Aldrich SML0006 Phusion TM High-Fidelity DNA Polymerase & dNTP Mix ThermoFisher Scientific F530N SuperScript III First-Strand Synthesis System for RT-PCR ThermoFisher Scientific 18080-051 Maxima SYBR Green/Flouorescein qPCR Master Mix ThermoFisher Scientific K0243 Gateway® BP Clonase TM II Enzyme Mix ThermoFisher Scientific 11789-020 18 of 26 The EMBO Journal 42:e111885 |2023 Ó2023 The Authors The EMBO Journal Chen Liu et al Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. Reagents and Tools table (continued) Reagent/Resource Reference or Source Identifier or Catalog Number Gateway® LR Clonase® II Enzyme mix ThermoFisher Scientific 11791020 Colloidal blue staining ThermoFisher Scientific LC6025 ECL Prime Western Blotting Detection Reagent Cytiva GERPN2232 Software Cytoscape v3.4.0http://www.cytoscape.org Shannon et al (2003) FIJI https://fiji.sc/ Schindelin et al (2012) RootTrace http://www.plant-image-analysis.org/software/ roottrace French et al (2008) JACoP https://imagej.nih.gov/ij/plugins/track/jacop.html Bolte and Cordelieres (2006) Graphpad Prism v9https://www.graphpad.com/scientific-software/prism/ Commercial Adobe Photoshop 2023 https://www.adobe.com Commercial Search Tool for the Retrieval of Interacting Genes/Proteins https://string-db.org Freeware Rv4.4.2https://www.r-project.org Freeware Other In Fusion HD Cloning Kit Clonetech/Takara 638909 SuperSignal Western Blot Enhancer ThermoFisher Scientific 46641 SuperSignal TM West Femto Maximum Sensitivity Substrate ThermoFisher Scientific 34094 SP8LIGHTINING MODE CONFOCAL MICROSCOPE https://www.leica-microsystems.com Commercial Phire Plant Direct PCR Master Mix ThermoFisher Scientific F160S pENTR TM /D-TOPO TM Cloning Kit Thermo Fisher Scientific K240020 GeneJET Plasmid Miniprep Kit ThermoFisher Scientific K0503 GeneJET Gel Extraction Kit ThermoFisher Scientific K0692 Thermo Scientific TM Orbitrap Fusion TM Lumos TM Tribrid TM Mass Spectrometer https://www.thermofisher.com/order/catalog/product/ IQLAAEGAAPFADBMBHQ Commercial Methods and Protocols Plant materials All the plant lines used in this study were in the Arabidopsis Columbia-0 (Col-0) accession except the ones indicated below. Primers used for genotyping and cloning are described in Appendix Tables. Except for the Transfer (T)-DNA insertion mutants obtained from the NASC (The Nottingham Arabidopsis Stock Centre) as indicated in the Reagents and Tools table above, the following mutants and transgenic lines used in this study were described previously: dcp1-1 (Xu et al,2006), dcp1-3 (Martinez de Alba et al,2015), dcp2-1 (Chantarachot et al,2020), dcp5-1 (Xu & Chua, 2009), pat1-1 (Roux et al,2015), xrn4-5 (Souret et al,2004), brk1 (Dyachok et al,2008), scar1234 (Dyachok et al,2008), arpc5 (Pratap Sahi et al,2017), 35S:GFP-DCP1 (Gutierrez-Beltran et al,2015), 35Spro:DCP2-YFP (Jang et al,2019), dcp5-1 DCP5pro: DCP5-3HA (Xu & Chua, 2009), RH12pro:RH12-GFP (Jang et al,2019), DCP1pro:DCP1-GFP (Yu et al,2019), DCP1pro:DCP1 S237A -GFP and DCP1pro:DCP1 S237D -GFP (Yu et al,2019), SOK3pro:SOK3-YFP (van Dop et al,2020), SCAR2pro:SCAR2-mCherry (preprint: Chin et al,2020), BRK1pro:BRK1-mRuby (preprint: Chin et al,2020), BRK1pro:BRK1-YFP (Dyachok et al,2008), UBQ10pro:LifeActmCherry (preprint: Chin et al,2020), SPIpro:SPI-Ypet (preprint: Chin et al,2020), PIN2pro:PIN2-GFP (Xu & Scheres, 2005), PIN7pro:PIN7- GFP (Zhou et al,2010), DEX˃RAB-A2c DN and RAB-A5cpro:RAB-A5c (Kirchhelle et al,2016). In all experiments, plants from T1/F1 (colocalization experiments), T2/F2, or T3 (for physiological experiments) generations were used. Construction of the sok1 sok3 CRISPR deletion mutant EC1-driven SOK1 deletion constructs were made as follows: single guide RNAs (sgRNAs) were synthesized with respective overhangs. The two complementary oligos were annealed and inserted into pEN-2xChimera using BpiI and BsmBI restriction enzymes. The sgRNAs were then transferred into pUbiCAS9-Red (for protoplasts) or pEciCAS9-Red (for stable transformation in A. thaliana) by Gateway â single-site LR recombination-mediated cloning. The efficiency of the sgRNAs was tested in protoplasts: Arabidopsis mesophyll protoplasts for transient expression of the CRISPR/Cas9 construct (pUbiCAS9-Red) were prepared as described previously with minor modifications (Yoo et al,2007). Approximately, 80,000 protoplasts were transformed with 16 lg of plasmid (pUbiCAS9-Red) and incubated for 48 h at 22°C under long photoperiod conditions (150 lmol/m 2 /s and 16-h-light/8-h-dark cycles). Genomic DNA was isolated, and concentrations were adjusted before performing semi-quantitative PCR using oligonucleotides flanking the region Ó2023 The Authors The EMBO Journal 42:e111885 |2023 19 of 26 Chen Liu et al The EMBO Journal Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. targeted for deletion (Liu et al,2017; Labun et al,2019; Ursache et al,2021). To create the SOSEKI3 (AT2G28150) deletion mutant, we used a previously described multiplexed editing approach (Ursache et al,2021). We designed sgRNAs using the CRISPR-P 2.0 (http://crispr.hzau.edu.cn/CRISPR2; Liu et al,2017) and CHOPCHOP (https://chopchop.cbu.uib.no/; Labun et al,2019) web tools. To delete the complete coding sequence of the gene, we targeted two sites near the start codon of SOK3, and one site downstream of the stop codon, along with equivalent sgRNAs for SOK2 (AT5G10150). Following the published protocol, we cloned all sgRNAs into a single pRU292 destination vector. This vector allows for zCas9i expression under the UBi4.2 promoter, and expression of the sgRNAs and has a FastGreen selection marker. sgRNAs and oligonucleotide sequences used for cloning are shown in Source Table 1. The resulting binary vector was transformed into the sok1 mutant background using the floral dipping method. We selected green fluorescing seeds and screened the resulting seedlings for large deletions in SOK3 and the absence of large deletions in SOK2 using PCR. We validated the mutations in SOK3 by direct genespecific sequencing and crossed out the transgene by selecting nonfluorescent seeds in the T2 generation. We confirmed that SOK2 was not deleted in this line, but there were small deletions in this gene. In the T3 generation, we selected double homozygous sok1 sok3 plants. Plant growth conditions Arabidopsis seedlings were sterilized and germinated on halfstrength Murashige and Skoog (MS) agar medium under long-day conditions (16 h light/8 h dark). In all experiments involving the use of mutants or pharmacological treatments, the medium was supplemented with 1% (w/v) sucrose or as otherwise specified. Arabidopsis plants for crosses, phenotyping of the above-ground part, and seed collection were grown on soil in a plant chamber at 22°C/19°C, 14-h-light/10-h-dark or 16-h-light/8-h-dark cycles, and light intensity 150 lE/m 2 /s. Arabidopsis and Nicotiana benthamiana plants were grown in Aralab or Percival cabinets at 22°C, 16-h- light/8-h-dark cycles, and a light intensity of 150 lE/m 2 /s. Phenotypic analysis and drug treatments For quantification of phenotypes, seedlings were surface sterilized and grown on half-strength MS medium plates with 1% (w/v) sucrose. For a given genotype, differential contrast interference (DIC) images were captured on a Leica DM2500, Leica DM6B, or Leica DM6000. To define root length, images were captured from the plates using a Leica DM6000 with a motorized stage and computationally compiled together. Root length or size was determined using Image J/Fiji by comparing the measurement with the WT or mock conditions (National Institute of Health). The stock solutions of 50 mM biotin, 2 mM FM4-64, 2 mM dexamethasone, 10 mM cytochalasin D (Cyto D), 10 mM Amiprofos methyl (APM), 1 mM latrunculin B, 50 mM brefeldin A (BFA), 200 mM CK666 and 50 mM cycloheximide (CHX) were dissolved in dimethyl sulfoxide (DMSO), while 40 mM isoxaben was dissolved in ethanol. 1.0 mg/ ml Propidium iodide (PI) was in water. These inhibitors or drugs were diluted in half-strength MS medium with corresponding concentration and duration, and the final DMSO concentration was ≤0.1% (v/v) in all experimental analyses. Vertically grown 4- to 5- day-old Arabidopsis seedlings were incubated in a half-strength liquid MS medium containing the corresponding drugs for each specific time course treatment as indicated. Bacterial strains, cloning, and constructs Electrocompetent Agrobacterium (Agrobacterium tumefaciens) strain C58C1 Rif R (pMP90) or GV3101 Rif R (i.e., a cured nopaline strain commonly used for infiltration) was used for electroporation and Nicotiana benthamiana infiltration. The Goldengate-compatible TurboID vector (35Spro:sGFP-TurboID-HF) was previously described (Arora et al,2020). Cloning was done according to standard Goldengate cloning procedures. In brief, TurboID was synthesized with BsaI overhangs using the codon optimization tool of Integrated DNA Technologies for codon-optimized expression in Arabidopsis (Eurofins). The coding sequence of DCP1 was PCR amplified using Phusion TM High-Fidelity DNA Polymerase & dNTP Mix (Thermo Fisher Scientific, F530N) using the GFP-DCP1 vector as a template. The 35S promoter carrying level 0 vector (pICSL13002) and all PCR parts with BsaI overhands were ligated to the Level1/2 vectors pICSL86900 and pICSL86922. Other constructs of DCP1 were generated by Gateway cloning with pENTR-DCP1 into different destination vectors which have different tags in N or C termini as indicated in the text or legends. The coding sequence of ARPC5 was PCR amplified using Phusion TM High-Fidelity DNA Polymerase & dNTP Mix (Thermo Fisher Scientific, F530N) using the cDNA from 7-day- old seedling of WT plants with pENTR TM /D-TOPO TM Cloning Kit (Thermo Fisher Scientific, K240020). For cloning, the bacterial strain NEB10 (New England Biolabs #C3019H) or NEB stable (New England Biolabs C3040H; CRISPR constructs) was used. For BiFC and colocalization constructs, At1g33050, At2g26920, ECT4,ECT6, MLN51,FLXL1,EIN2,VAP27-1, and At5g53330 were generated by Gateway cloning. pENTR vectors were generated via BP reaction with pDONR221 (Invitrogen) and PCR product amplicons from RTPCR using cDNA from 1-week-old seedlings. As destination vectors, pSITE17 (nYFP-GW), pSITE18 (cYFP-GW), and pG5 vector (custommade Gateway vector with tagRFP) were used. DCP1 antibody production The DCP1 cDNA in pGAT4 (hexahistidine-tagged vector) was transformed in BL21 (DE3) Rosetta or BL21 (DE3) Rosetta II Escherichia coli cells. Bacterial cultures were grown in 800 ml of Luria Bertani (LB) medium supplemented with 100 mg/l of ampicillin and 25 mg/l of chloramphenicol. Protein production was induced at OD 600 =0.5 with 0.05 to 1 mM isopropyl b-D-1- thiogalactopyranoside (IPTG). After 3 h, the cells were harvested by centrifugation at 2,500 gfor 20 min at room temperature and frozen overnight at 80°C. Preparation of hexahistidine-tagged recombinant DCP1 was performed according to the manufacturer’s instructions in Tris-buffer (Qiagen, 30210). The abundance of proteins was estimated by coomassie brilliant blue (CBB) staining by SDS-PAGE or on immunoblots using a-his. The DCP1 protein was dialyzed overnight in assay buffer (2 l) and was used to immunize four mice. The antibodies were further purified by solid-phase absorption using columns with DCP1. Pre-immune sera were also collected and used as an additional negative control, producing no signal. Immunoblotting Infiltrated N. benthamiana leaves or Arabidopsis leaves and seedlings were harvested, and their proteins were extracted. The tissue 20 of 26 The EMBO Journal 42:e111885 |2023 Ó2023 The Authors The EMBO Journal Chen Liu et al Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. samples were flash-frozen in liquid Ν 2 and kept at 80°C until further processing. The samples were crushed using a liquid Ν 2 - cooled mortar and pestle, and the crushed material was transferred to a 1.5-ml or 15-ml tube. Extraction buffer (EB; 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% [v/v] glycerol, 2 mM ethylenediamine tetra acetic acid [EDTA], 5 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], Protease Inhibitor Cocktail [Sigma- Aldrich, P9599] and 0.5% [v/v] IGEPAL CA-630 [Sigma-Aldrich]) was added according to the plant material used. The lysates were pre-cleared by centrifugation at 16,000 gat 4°C for 15 min, and the supernatant was transferred to a new 1.5-ml tube. This step was repeated two times and the protein concentration was determined by the RC DC Protein Assay Kit II (Bio-Rad, 5000122). Two times Laemmli buffer was added, and equivalent amounts of protein (~30 lg) were separated by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 1.0 mm thick 4–12% [w/v] gradient polyacrylamide Criterion Bio-Rad) in 3-(N-Morpholino) propane sulfonic acid (MOPS) buffer (Bio-Rad) at 150 V. Subsequently, proteins were transferred onto a polyvinylidene fluoride (PVDF; Bio-Rad) membrane with 0.22-lm pore size. The membrane was blocked with 3% (w/v) BSA fraction V (Thermo Fisher Scientific) in phosphate buffered saline-Tween 20 (PBS-T) for 1 h at room temperature (RT), followed by incubation with horseradish peroxidase (HRP)-conjugated primary antibody at RT for 2 h (or primary antibody at RT for 2 h and corresponding secondary antibody at RT for 2 h). The following antibodies were used: streptavidin-HRP (Sigma-Aldrich; 1:25,000, N100), mouse a-FLAG-HRP (Sigma- Aldrich, A8592, 1:2,000), rat a-tubulin (Santa Cruz Biotechnology, 1:1,000), rabbit a-GFP (Millipore, AB10145, 1:10,000), mouse a-RFP (Agrisera, AS15 3028, 1:5,000), a-mouse (Amersham ECL Mouse IgG, HRP-linked whole Ab [from sheep], NA931, 1:10,000), a-rabbit (Amersham ECL Rabbit IgG, HRP-linked whole Ab [from donkey], NA934, 1:10,000), a-rat (IRDyeâ800 CW Goat a-Rat IgG [H +L], LI-COR, 925-32219, 1:10,000), and a-rabbit (IRDye â800 CW Goat a-Rabbit IgG, LI-COR, 926-3221, 1:10,000). Chemiluminescence was detected with the ECL Prime Western Blotting Detection Reagent (Cytiva, GERPN2232) and SuperSignal TM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34094). The bands were visualized using an Odyssey infrared imaging system (LICOR). APEAL approach details After 24 h treatment (syringe infiltration) with 50 lM biotin (diluted in 10 mM MgCl 2 , 10 mM MES pH 5.7), 2.5 ml pulverized Arabidopsis leaves or seedlings (~0.5 g FW) were extracted in 5 ml EB (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% [v/v] glycerol, 0.5 mM EDTA, 1 mM DTT, 0.5% [v/v] IGEPAL CA-630 [Sigma- Aldrich] and Protease inhibitor Cocktail [1:100 dilution, Sigma- Aldrich, P9599]). Extracts were incubated on a shaker at 4°C for 10 min and then centrifuged at 4°C, 13,000 gfor 30 min. Five milliliters of clarified supernatants were incubated with 100 ll magnetic FLAG beads (Sigma-Aldrich, A8592) at 4°C for 2 h with gentle rotation, then the FLAG beads were precipitated with DynaMag TM -2 Magnet (Thermo Fisher Scientific, 112321D) and washed four times with 1 ml EB (5 min each time). The supernatants (“flowthrough1”) were filtered through PD-10 columns (Cytiva, 17085101), then the biotin-depleted “flow-though2” was incubated with 100 ll Dynabeads TM M-280 Streptavidin (Thermo Fisher Scientific, 11205D) at 4°C for 2 h with gentle rotation. The FLAG beads and Dynabeads were washed five times with 1 ml EB and eight times with 1 ml 50 mM NH 4 HCO 3 (5 min each time). The beads were then collected on a magnetic rack and subjected to onbeads digestion followed by mass spectrometry. For immunoblot analysis, the proteins were eluted from the FLAG beads with 60 ll 2×Laemmli buffer (Bio-Rad, 1610747) supplemented with 10 mM DTT and incubated at 95°C for 10 min. The proteins were eluted from the Dynabeads with 60 ll2×Laemmli buffer supplemented with 5 mM biotin, 2% (w/v) SDS, and 10 mM DTT and incubated at 95°C for 20 min. On-beads digestion After immunoprecipitation and extensive washing with 25 mM NH 4 HCO 3 , 0.1 lg trypsin in 10 ll of 2 mM CaCl 2 , 10% (v/v) acetonitrile, and 25 mM NH 4 HCO 3 was added to beads and incubated at 37°C overnight. Fresh trypsin (0.1 lg) in 10 ll25mM NH 4 HCO 3 was added to beads and incubated for another 4 h. The digested supernatant was transferred into a clean centrifuge tube, and acetonitrile was evaporated under a vacuum. The samples were then acidified and desalted using a C18 stage tip before being analyzed by nano-liquid chromatography–tandem mass spectrometry. Liquid chromatography–tandem mass spectrometry Samples were analyzed by LC–MS using a Nano LC–MS/MS apparatus (Dionex Ultimate 3000 RLSCnano System) interfaced with an Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). Samples were loaded onto a fused silica trap column (Acclaim PepMap 100, 75 lm×2 cm; Thermo Fisher Scientific). After washing for 5 min at 5 ll/min with 0.1% (v/v) trifluoroacetic acid (TFA), the trap column was brought in-line with an analytical column (Nanoease MZ peptide BEH C18, 130 A, 1.7 lm, 75 lm×250 mm, Waters) for LC–MS/MS. Peptides were fractionated at 300 nl/min using a segmented linear gradient 4–15% B in 30 min (where A: 0.2% formic acid, and B: 0.16% formic acid, 80% acetonitrile), 15– 25% B in 40 min, 25–50% B in 44 min, and 50–90% B in 11 min. Solution B was then returned to 4% for 5 min for the next run. The scan sequence began with an MS1 spectrum (Orbitrap analysis, resolution 120,000, scan range from m/z 350–1,600, automatic gain control (AGC) target 1E6, maximum injection time 100 ms). The top S (3 s) and dynamic exclusion of 60 s were used for the selection of parent ions for MS/MS. Parent masses were isolated in the quadrupole with an isolation window of 1.4 m/z, automatic gain control (AGC) target 1E5, and fragmented with higher-energy collisional dissociation with a normalized collision energy of 30%. The fragments were scanned in Orbitrap with a resolution of 30,000. The MS/MS scan range was determined by the charge state of the parent ion, but the lower limit was set at 100 amu. Database search LC–MS/MS data were analyzed with Maxquant (version 1.6.10.43) with the Andromeda search engine. The type of LC–MS run was set to 1 (label-free). LC–MS data were searched against The Arabidopsis Information Resource (TAIR) plus a common contaminant database. Protease was set as trypsin, which allowed two miscuts. N-terminal acetylation and oxidation at methionine were set as variable modifications. Maximum two variable modification was allowed. Protein Ó2023 The Authors The EMBO Journal 42:e111885 |2023 21 of 26 Chen Liu et al The EMBO Journal Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. and peptide false discovery rate (FDR) were set to 1%. Reverse hit and common contaminants, as well as proteins identified only by modified sites, were removed. GO term analysis was performed using a combination of the Panther database and BioConductor package in R (Bonnot et al.2019). Visualization of networks and analyses Cytoscape 3.5.1 was used. Tab-delimited files containing the input data were uploaded. Unless otherwise indicated, the default layout was an edge-weighted spring-embedded layout, with NormSpec used as edge weight. Nodes were manually re-arranged from this layout to increase visibility and highlight specific proximity interactions. The layout was exported as a PDF and eventually converted to a. TIFF file with Lempel–Ziv–Welch (common name LZW) compression. Agrobacterium-mediated transient transformation of Nicotiana benthamiana N. benthamiana plants were grown under normal light and dark regimes at 25°C and 70% relative humidity. Three- to four-week-old N. benthamiana plants were watered from the bottom ~2 h before infiltration. Transformed Agrobacterium strain C58C1 Rif R (pMP90) or GV3101 Rif R harboring the constructs of interest were used to infiltrate N. benthamiana leaves and for transient expression of binary constructs by Agrobacterium-mediated transient infiltration of lower epidermal leaf cells. Transformed Agrobacterium colonies were grown for ~20 h in a shaking incubator (200 rpm) at 28°Cin 5 ml of yeast extract broth (YEB) medium (5 g/l beef extract, 1 g/l yeast extract, 5 g/l peptone, 0.5 g/l MgCl 2 , and 15 g/l bacterial agar), supplemented with appropriate antibiotics (i.e., 100 g/l spectinomycin). After incubation, the bacterial culture was transferred to 15-ml Falcon tubes and centrifuged (10 min, 5,000 g). The pellets were washed with 5 ml of infiltration buffer (10 mM MgCl 2 , 10 mM MES pH 5.7), and the final pellet was resuspended in infiltration buffer supplemented with 100 lM acetosyringone from a stock diluted in dimethyl formamide. The bacterial suspension was diluted with infiltration buffer to adjust the inoculum cell density to a final OD 600 value of 0.2–1.0. The inoculum was incubated for 2 h at room temperature before infiltration into N. benthamiana leaves by gentle pressure infiltration of the lower epidermis of leaves (fourth and older true leaves were used, and about 4/5-1/1 of their full size) with a 1-ml hypodermic syringe without a needle. Bimolecular fluorescence complementation (BiFC) The BiFC assay was done in N. benthamiana plants. Excitation wavelengths and emission filters were 514 nm/band-pass 530– 550 nm for YFP and 488 nm/band-pass 650–710 nm for chloroplast auto-fluorescence. The objective used was a HC PL APO 40×/1,30 oil CS2 with NA =1.3 (Leica SP8 confocal system). TIRFM imaging and tracking analyses TIRF microscopy images were acquired using MetaMorph software on an Olympus IX-81 microscope. A DV2 image splitter (MAG Biosystems) was used to separate GFP and RFP emission signals. Time-lapse movies were obtained at 100-ms intervals. For MSD analysis, 30-s-long movies with 100-ms intervals and 200-ms exposure were used. Particle tracking was limited to the amount of time that the plasma membrane remained at the focal plane; the median track length was 2,000 frames, corresponding to 3.5 s of imaging. The tracking of particles was performed with the Mosaic suite of Fiji or NanoTrackJ/TrackMate, using the following parameters: radius 3 of fluorescence intensity, a link range of 1, cutoff of 0.1%, and a maximum displacement of 8 px, assuming Brownian dynamics. Quantification of fluorescent intensity, FRAP, and FRET To create the most comparable lines to measure the fluorescence intensity of reporters in multiple mutant backgrounds, we crossed homozygous mutant plants carrying the marker with either a wildtype plant (to yield progeny heterozygous for the recessive mutant alleles and the reporter) or crossed to a mutant only plant (to yield progeny homozygous for the recessive mutant alleles and heterozygous for the reporter). Fluorescence was measured as a mean integrated density in regions of interest (ROIs) with the subtraction of the background (a proximal region that was unbleached and had less signal intensity than the signal of the ROI region). FRAP mode of Zeiss 780 ZEN software was set up for the acquisition of 3 prebleach images, 1 bleach scan, and 96 post-bleach scans (or more). Bleaching was performed using the 488-, 514-, and 561-nm laser lines at 100% transmittance and 20–40 iterations depending on the region and the axial resolution (iterations increased in deeper tissues to compensate for the increased light scattering). In FRAP the width of the bleached ROI was set to 2–10 lm. Pre- and postbleach scans were at minimum possible laser power (0.8% transmittance) for 458 nm or 514 nm (4.7%) and 5% for 561 nm; 512 ×512 8-bit pixel format; pinhole of 181 lm(>2 Airy units) and zoom factor of 2.0. The background values were subtracted from the fluorescence recovery values, and the resulting values were normalized by the first post-bleach time point and divided by the maximum fluorescent time-point set maximum intensity as 1. The objective used was a plan-apochromat 40×with NA =1.2 M27 (Zeiss). The following settings were used for photobleaching DCP1: 10–20 iterations for DCP1-GFP; 10 to 60 s per frame; 100% transmittance with the 458- to 514-nm laser lines of an argon laser. Pre- and post-bleach scans were at minimum possible laser power (1.4 to 20% transmittance) for the 488-nm and 0% for all other laser lines, 512 ×512- pixel format, and zoom factor of 5.1. The fluorescence intensity recovery values were determined, the background values were subtracted from the fluorescence recovery values, and the resulting values were normalized against the first post-bleach time point. SEFRET analyses were conducted using the method described previously (preprint: Liu et al,2022) and photoacceptor FRET was conducted with the method as described (Karpova et al,2003). Image analyses and intensity measurements (Integrated Density) were done using Fiji v. 1.52 software (rsb.info.nih.gov/ij). For relative values, the intensities were normalized against the background signal intensity in raw images/micrographs. The calculations were done with arbitrary units (denoted as AUs). The dwell time rate of tagged proteins in FRAP experiments was calculated by the single exponential fit (Moschou et al,2016). Colocalization was analyzed using Pearson statistics (Spearman or Manders analyses produced similar results or trends, with Fiji, coloc2 tool; French et al,2008). Images were prepared in Adobe Photoshop version 2023. Time series movies were compressed, corrected, and exported as .avi extension files. The nonspecific fluorescence decay was corrected using Fiji and default options using the bleaching correction tool. Videos were digitally enhanced with Fiji-implemented filters, 22 of 26 The EMBO Journal 42:e111885 |2023 Ó2023 The Authors The EMBO Journal Chen Liu et al Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. correcting noise using the Gaussian blur option and pixel width set to 1.0. Immunocytochemistry, PLA, and imaging Immunocytochemistry was done as described previously (Moschou et al,2016). The primary antibody used was rabbit a-PAT1 (diluted 1:500; Roux et al,2015), rat a-tubulin YL1/2 (1:200; Santa Cruz Biotechnology), mouse a-DCP1 (diluted 1:100; prepared herein), and a-HA (diluted 1:300; Sigma-Aldrich). In brief, specimens were washed three times for 90 min in PBS-T and incubated overnight with anti-rabbit fluorescein isothiocyanate-conjugated (FITC) secondary antibody (Jackson ImmunoResearch, 711-095-152) diluted 1:200–250, DyLight TM 549 AffiniPure Fab Fragment Rabbit Anti-Mouse IgG (H +L; Jackson ImmunoResearch, 315-507-003), Rhodamine Red TM -X (RRX) AffiniPure Donkey Anti-Rabbit IgG (H +L; Jackson ImmunoResearch, 711-295-152), Tetramethylrhodamine (TRITC) Donkey Anti-Rabbit IgG (H +L; Jackson ImmunoR- esearch, 711-026-152), Alexa Fluorâ647 AffiniPure Donkey Anti- Mouse IgG (H +L; Jackson ImmunoResearch, 715-605-151) diluted 1:200–250. After washing in PBS-T and incubation with or without DAPI (1 lg/ml), specimens were mounted in Vectashield (Vector Laboratories) medium and observed within 48 h. Root tips were imaged using a Zeiss 780 confocal laser scanning microscope. The objective used was a plan-Apochromat 63×/1.4 oil immersion M27 (Zeiss) or an HC PL APO 40×/1.3 oil CS2 with NA =1.3 (Leica SP8 confocal system). Immunostaining of actin was done as described previously with minor modifications (Dyachok et al,2008). Roots of 5-day-old Arabidopsis seedlings were fixed for 1 h in actin-fixation buffer (50 mM PIPES, pH 7.2, with 20 mM EGTA and 20 mM MgSO 4 containing 2% [w/v] paraformaldehyde, 0.1% [v/v] Triton X-100 and 400 mM maleimidobenzoyl-N-hydroxy succinimide ester [Thermo Fisher Scientific, 22311]); the cell wall was digested for 30 min (in 0.2% [w/v] driselase and 0.15% [w/v] macerozyme). Then samples were incubated for 30 min in blocking buffer containing 2% (w/v) BSA and then incubated with Alexa Fluor 488 phalloidin (1:400 dilution, Thermo Fisher Scientific, A12379) overnight at 4°C. After washing with PBST buffer three times, samples were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and root tips were imaged using a Zeiss 780 confocal laser scanning microscope. The objective used was a plan-Apochromat 63×/1.4 oil immersion M27 (Zeiss). PLA immunolocalization was done as described previously (Alam, 2018; preprint: Liu et al,2022). Primary antibody combinations diluted 1:200 for a-GFP mouse (Sigma-Aldrich, SAB2702197), 1:200 for a-FLAG mouse (Sigma-Aldrich, F1804), 1:200 for a-RFP mouse (Agrisera, AS15 3028), and 1:200 for a-GFP rabbit (Millipore, AB10145) were used for overnight incubation at 4°C. Roots were then washed with microtubules-stabilizing buffer (MTSB: 50 mM PIPES, 5 mM EGTA, 2 mM MgSO 4 , 0.1% [v/v] Triton X-100) and incubated at 37°C for 3 h either with a-mouse plus and a-rabbit minus for PLA assay (Sigma-Aldrich, Duolink). PLA samples were then washed with MTSB and incubated for 3 h at 37°C with ligase solution. Roots were then washed 2×with buffer A (Sigma-Aldrich, Duolink) and treated for 4 h at 37°C in a polymerase solution containing fluorescent nucleotides as described (Sigma-Aldrich, Duolink). Samples were then washed 2×with buffer B (Sigma- Aldrich, Duolink), with 1% (v/v) buffer B for another 5 min, and then the specimens were mounted in Vectashield (Vector Laboratories) medium. Statistics The numerical data used in this publication are provided in Source Data as raw .csv files with the corresponding headings. Statistical analyses were performed in GraphPad (https://graphpad.com/)orR studio (R-project.org). Graphs were also generated by GraphPad Prism or R. PCC is calculated via Fiji, coloc2 tool. All statistical data show the mean s.d. (box plots) or the distribution of values (violin plots, kernel density) of at least three biologically independent experiments or samples, or as otherwise stated. Individual data points are on the plots. For violin plots, datasets were smoothed using heavy smoothing which gives a better idea of the overall distribution. In captions, Ndenotes biological replicates, and “n” technical replicates or population size. Each data set was tested whether it followed normal distribution when N≥3 by using the Shapiro normality test. The significance threshold was set at P<0.05 (significance claim), and the exact Pvalues are shown in the graphs or as asterisks. Details of the statistical tests applied, including the choice of the statistical method, are indicated in the corresponding figure caption. In boxplots or violin plots, upper and lower box boundaries, or lines in the violin plots when visible, represent the first and third quantiles, respectively, horizontal lines mark the median and whiskers mark the highest and lowest values. For regression analyses, the confidence intervals were calculated and are also shown (95% confidence band) of the best-fit line. For Wilcoxon, Pvalues are two-tailed, for Kruskal–Wallis Pvalues are approximate, for Welch-ANOVA or Welch–Forsythe in multiple comparisons, Pvalues are adjusted. For Kolmogorov–Smirnoff, P values are approximate. To increase the robustness of analyses when the normality was marginal (judged by the P), sometimes more than one test was used as indicated in the caption (data analyses). For acceptor photobleaching to determine the FRET-efficiency, FDR corrections were done through two-stage step-up method of Benjamini, Krieger, and Yekutieli. In one crucial experiment (dcp1- 3/SCAR2 localization), the researcher did not know the genotype, which was determined later (blind experiment). In all other cases, the experiments were not blind. Data availability The data generated or analyzed during this study are included in this published article. Additional information and materials generated for and/or reported in this article are available from the corresponding author upon request. The mass spectrometry proteomics data from this publication have been deposited to the ProteomeX- change Consortium via the PRIDE https://www.ebi.ac.uk/pride/ partner repository (Perez-Riverol et al,2022) with the dataset identifier PXD037701 (https://www.ebi.ac.uk/pride/archive/projects/ PXD037701). Expanded View for this article is available online. Acknowledgements The authors thank Sabrina Chin, Laurie Smith, Elison Blancaflor, Sebastian Y. Bednarek, Morten Petersen, Ping He, Annie Marion-Poll, Shu-Hsing Wu, Jan Ó2023 The Authors The EMBO Journal 42:e111885 |2023 23 of 26 Chen Liu et al The EMBO Journal Downloaded from https://www.embopress.org on July 3, 2024 from IP 150.214.182.235. Petr a sek, Kate rina  Slajcherov a, and Charlotte Kirchhelle for kindly providing published materials. This work was supported by the EU Marie Curie-RISE (“PANTHEON,”project number 872969; PNM), the Carl Tryggers Foundation (contract number 15 and 17:336; PNM), and partly by the Vetenskapsr adet (VR) research council (contract number 21679000; PNM), FORMAS research council (contract number 22924-000; PNM), Helge Ax:son Johnsons stiftelse (contract number F19-0419; CL), Hellenic Foundation of Research and Innovation (HFRI, contract number 06526; AM), IMBB-FORTH start-up funding (PNM), European Research Council (ERC; ‘DIRNDL’, contract number 833867; DW) and Spanish grant PID2020-119737GA-I00 funded by MCIN/AEI/437 (contract number 10.13039/501100011033; EGB). Author contributions Chen Liu: Data curation; formal analysis; funding acquisition; investigation; visualization; methodology; writing –review and editing. Andriani Mentzelopoulou: Data curation; software; formal analysis; investigation; visualization; writing –review and editing. Amna Muhammad: Investigation. Andriy Volkov: Resources; formal analysis; investigation; writing –review and editing. Dolf Weijers: Resources; funding acquisition. Emilio Gutierrez-Beltran: Resources; formal analysis; funding acquisition. Panagiotis N Moschou: Conceptualization; data curation; formal analysis; supervision; funding acquisition; investigation; visualization; methodology; writing –original draft; project administration; writing –review and editing. Disclosure and competing interests statement The authors declare that they have no conflict of interest. References Alam MS (2018) Proximity ligation assay (PLA). 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