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Plasmodesmata (PD) are plant-specific membrane-lined channels that create cytoplasmic and membrane continuities between adjacent cells, thereby facilitating cell–cell communication and virus movement. Plant cells have evolved diverse mechanisms to regulate PD plasticity in response to numerous environmental stimuli. In particular, during defense against plant pathogens, the defense hormone, salicylic acid (SA), plays a crucial role in the regulation of PD permeability in a callose-dependent manner. Here,we uncover a mechanism by which plants restrict the spreading of virus and PD cargoes using SA signaling by increasing lipid order and closure of PD. We showed that exogenous SA application triggered the compartmentalization of lipid raft nanodomains through a modulation of the lipid raft-regulatory protein, Remorin (REM). Genetic studies, superresolution imaging, and transmission electron microscopy observation together demonstrated that Arabidopsis REM1.2 and REM1.3 are crucial for plasma membrane nanodomain assembly to control PD aperture and functionality. In addition, we also found that a 14-3-3 epsilon protein modulates REM clustering and membrane nanodomain compartmentalization through its direct interaction with REM proteins. This study unveils a molecular mechanism by which the key plant defense hormone, SA, triggers membrane lipid nanodomain reorganization, thereby regulating PD closure to impede virus spreading.

Plasmodesmata (PD) are plasma membrane-lined pores that connect neighbouring plant cells, bridging the cell wall and establishing cytoplasmic and membrane continuity between cells. PD are dynamic structures regulated by callose deposition in a variety of stress and developmental contexts. This process crudely controls the aperture of the pore and thus the flux of molecules between cells. During pathogen infection, plant cells initiate a range of immune responses and it was recently identified that, following perception of fungal and bacterial pathogens, plant cells initially close their PD. Systemic defence responses depend on the spread of signals between cells, raising questions about whether PD are in different functional states during different immune responses. It is well established that viral pathogens exploit PD to spread between cells, but it has more recently been identified that protein effectors secreted by fungal pathogens can spread between host cells via PD. It is possible that many classes of pathogens specifically target PD to aid infection, which would infer antagonistic regulation of PD by host and pathogen. How PD regulation benefits both host immune responses and pathogen infection is an important question and demands that we examine the multicellular nature of plant–pathogen interactions.

Plasmodesmata are intercellular pores connecting together most plant cells. These structures consist of a central constricted form of the endoplasmic reticulum, encircled by some cytoplasmic space, in turn delimited by the plasma membrane, itself ultimately surrounded by the cell wall. The presence and structure of plasmodesmata create multiple routes for intercellular trafficking of a large spectrum of molecules (encompassing RNAs, proteins, hormones and metabolites) and also enable local signalling events. Movement across plasmodesmata is finely controlled in order to balance processes requiring communication with those necessitating symplastic isolation. Here, we describe the identities and roles of the molecular components (specific sets of lipids, proteins and wall polysaccharides) that shape and define plasmodesmata structural and functional domains. We highlight the extensive and dynamic interactions that exist between the plasma/endoplasmic reticulum membranes, cytoplasm and cell wall domains, binding them together to effectively define plasmodesmata shapes and purposes.

Significance Cell–cell communication is a prerequisite of multicellular development and noncell autonomous stem cell induction has been conserved during evolution. Cytoplasmic bridges, called plasmodesmata, which facilitate the exchange of molecules between neighboring cells, are a striking innovation for cell–cell signaling in plants. Here, we show that plasmodesmata function is required for the activity of shoot apical stem cells in Arabidopsis and provide evidence that the stem cell inducing transcription factor WUSCHEL moves from the niche into the stem cells via this route. WUSCHEL movement is functionally relevant and mediated by multiple protein domains. Because parts of the protein that restrict movement are required for homodimerization, the formation of WUSCHEL dimers might contribute to the regulation of stem cell activity in Arabidopsis . Cell–cell communication is essential for multicellular development and, consequently, evolution has brought about an array of distinct mechanisms serving this purpose. Consistently, induction and maintenance of stem cell fate by noncell autonomous signals is a feature shared by many organisms and may depend on secreted factors, direct cell–cell contact, matrix interactions, or a combination of these mechanisms. Although many basic cellular processes are well conserved between animals and plants, cell-to-cell signaling is one function where substantial diversity has arisen between the two kingdoms of life. One of the most striking differences is the presence of cytoplasmic bridges, called plasmodesmata, which facilitate the exchange of molecules between neighboring plant cells and provide a unique route for cell–cell communication in the plant lineage. Here, we provide evidence that the stem cell inducing transcription factor WUSCHEL (WUS), expressed in the niche, moves to the stem cells via plasmodesmata in a highly regulated fashion and that this movement is required for WUS function and, thus, stem cell activity in Arabidopsis thaliana . We show that cell context-independent mobility is encoded in the WUS protein sequence and mediated by multiple domains. Finally, we demonstrate that parts of the protein that restrict movement are required for WUS homodimerization, suggesting that formation of WUS dimers might contribute to the regulation of apical stem cell activity.

Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has beenmade in identifying PD proteins, the role played by majormembrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of “native” PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositolanchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callosemodifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes.

The coordinated redistribution of sugars from mature “source” leaves to developing “sink” leaves requires tight regulation of sugar transport between cells via plasmodesmata (PD). Although fundamental to plant physiology, the mechanisms that control PD transport and thereby support development of new leaves have remained elusive. From a forward genetic screen for altered PD transport, we discovered that the conserved eukaryotic glucose- TOR (TARGET OF RAPAMYCIN) metabolic signaling network restricts PD transport in leaves. Genetic approaches and chemical or physiological treatments to either promote or disrupt TOR activity demonstrate that glucose-activated TOR decreases PD transport in leaves. We further found that TOR is significantly more active in mature leaves photosynthesizing excess sugars than in young, growing leaves, and that this increase in TOR activity correlates with decreased rates of PD transport. We conclude that leaf cells regulate PD trafficking in response to changing carbohydrate availability monitored by the TOR pathway.

Plants sense microbial signatures via activation of pattern recognition receptors (PPRs), which trigger a range of cellular defences. One response is the closure of plasmodesmata,which reduces symplastic connectivity and the capacity for direct molecular exchange between host cells. Plasmodesmal flux is regulated by a variety of environmental cues but the downstream signalling pathways are poorly defined, especially the way in which calcium regulates plasmodesmal closure. Here, we identify that closure of plasmodesmata in response to bacterial flagellin, but not fungal chitin, is mediated by a plasmodesmal-localized Ca2+-binding protein Calmodulin-like 41 (CML41). CML41 is transcriptionally upregulated by flg22 and facilitates rapid callose deposition at plasmodesmata following flg22 treatment. CML41 acts independently of other defence responses triggered by flg22 perception and reduces bacterial infection. We propose that CML41 enables Ca2+-signalling specificity during bacterial pathogen attack and is required for a complete defence response against Pseudomonas syringae.

In plants, mounting an effective innate immune strategy against microbial pathogens involves triggering local cell death within infected cells as well as boosting the immunity of the uninfected neighboring and systemically located cells. Although not much is known about this, it is evident that well-coordinated cell—cell signaling is critical in this process to confine infection to local tissue while allowing for the spread of systemic immune signals throughout the whole plant. In support of this notion, direct cell-to-cell communication was recently found to play a crucial role in plant defense. Here, we provide experimental evidence that salicylic acid (SA) is a critical hormonal signal that regulates cell-to-cell permeability during innate immune responses elicited by virulent bacterial infection in Arabidopsis thaliana. We show that direct exogenous application of SA or bacterial infection suppresses cell—cell coupling and that SA pathway mutants are impaired in this response. The SA- or infection-induced suppression of cell—cell coupling requires an ENHANCED DESEASE RESISTANCE1— and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1—dependent SA pathway in conjunction with the regulator of plasmodesmal gating PLASMODESMATA-LOCATED PROTEIN5. We discuss a model wherein the SA signaling pathway and plasmodesmata-mediated cell-to-cell communication converge under an intricate regulatory loop.

• Synaptotagmin 1 (SYT1) has been recognised as a tethering factor of plant endoplasmic reticulum (ER)–plasma membrane (PM) contact sites (EPCSs) and partially localises to around plasmodesmata (PD). However, other components of EPCSs associated with SYT1 and functional links between the EPCSs and PD have not been identified. • We explored interactors of SYT1 by immunoprecipitation and mass analysis. The dynamics, morphology and spatial arrangement of the ER in Arabidopsis mutants lacking the EPCS components were investigated using confocal microscopy and electron microscopy. PD permeability of EPCS mutants was assessed using a virus movement protein and free green fluorescent protein (GFP) as indicators. • We identified two additional components of the EPCSs, SYT5 and SYT7, that interact with SYT1. The mutants of the three SYTs were defective in the anchoring of the ER to the PM. The ER near the PD entrance appeared to be weakly squeezed in the triple mutant compared with the wild-type. The triple mutant suppressed cell-to-cell movement of the virus movement protein, but not GFP diffusion. • We revealed major additional components of EPCS associated with SYT1 and suggested that the EPCSs arranged around the PD squeeze the ER to regulate active transport via PD.

The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane.