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Pathogenesis-related (PR) proteins are induced by abiotic and biotic stresses and generally considered as part of the plant defense mechanism. However, it remains yet largely unclear if and how they are involved in virus infection. Our recent quantitative, comparative proteomic study identified three PR-5 family proteins that are significantly differentially accumulated in the plasmodesmata (PD)-enriched fraction isolated from Nicotiana benthamiana leaves infected by turnip mosaic virus (TuMV). In this study, we employed the TuMV-Arabidopsis pathosystem to characterize the involvement of two Arabidopsis orthologs, AtOSM34 and AtOLP of the three N. benthamiana PR-5-like proteins. We show that AtOSM34 and AtOLP are PD-localized proteins and their expression is up- and downregulated in response to TuMV infection, respectively. Deficiency or overexpression of AtOLP does not affect viral RNA accumulation. Knockdown of AtOSM34 inhibits TuMV infection, whereas its overexpression promotes viral infection. We further demonstrate that AtOSM34 functions as a proviral factor through diminishing PD callose deposition to promote viral intercellular movement, targeting the viral replication complex to enhance viral replication, and suppressing the ROS-mediated antiviral response. Taken together, these data suggest that TuMV has evolved the ability to selectively upregulate and subvert AtOSM34, a PR-5 family protein to assist its infection., (© 2024 His Majesty the King in Right of Canada. New Phytologist © 2024 New Phytologist Foundation. Reproduced with the permission of the Minister of Agricultural and Agri‐Food Canada.)

Plasmodesmata are plasma membrane-lined connections that join plant cells to their neighbours, establishing an intercellular cytoplasmic continuum through which molecules can travel between cells, tissues, and organs. As plasmodesmata connect almost all cells in plants, their molecular traffic carries information and resources across a range of scales, but dynamic control of plasmodesmal aperture can change the possible domains of molecular exchange under different conditions. Plasmodesmal aperture is controlled by specialised signalling cascades accommodated in spatially discrete membrane and cell wall domains. Thus, the composition of plasmodesmata defines their capacity for molecular trafficking. Further, their shape and density can likewise define trafficking capacity, with the cell walls between different cell types hosting different numbers and forms of plasmodesmata to drive molecular flux in physiologically important directions. The molecular traffic that travels through plasmodesmata ranges from small metabolites through to proteins, and possibly even larger mRNAs. Smaller molecules are transmitted between cells via passive mechanisms but how larger molecules are efficiently trafficked through plasmodesmata remains a key question in plasmodesmal biology. How plasmodesmata are formed, the shape they take, what they are made of, and what passes through them regulate molecular traffic through plants, underpinning a wide range of plant physiology., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

In leaves of C 4 plants, the reactions of photosynthesis become restricted between two compartments. Typically, this allows accumulation of C 4 acids in mesophyll (M) cells and subsequent decarboxylation in the bundle sheath (BS). In C 4 grasses, proliferation of plasmodesmata between these cell types is thought to increase cell-to-cell connectivity to allow efficient metabolite movement. However, it is not known whether C 4 dicotyledons also show this enhanced plasmodesmal connectivity and so whether this is a general requirement for C 4 photosynthesis is not clear. How M and BS cells in C 4 leaves become highly connected is also not known. We investigated these questions using 3D- and 2D-electron microscopy on the C 4 dicotyledon Gynandropsis gynandra as well as phylogenetically close C 3 relatives. The M-BS interface of C 4 G. gynandra showed higher plasmodesmal frequency compared with closely related C 3 species. Formation of these plasmodesmata was induced by light. Pharmacological agents that perturbed photosynthesis reduced the number of plasmodesmata, but this inhibitory effect could be reversed by the provision of exogenous sucrose. We conclude that enhanced formation of plasmodesmata between M and BS cells is wired to the induction of photosynthesis in C 4 G. gynandra., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Sugar loading of developing seeds comprises a cohort of transport events that contribute to reproductive success and seed yield. Understanding these events is most advanced for grain crops (Brassicaceae, Fabaceae and Gramineae) and Arabidopsis. For these species, 75-80% of their final seed biomass is derived from phloem-imported sucrose. Sugar loading consecutively traverses three genomically distinct, and symplasmically isolated, seed domains: maternal pericarp/seed coat, filial endosperm and filial embryo. Sink status of each domain co-ordinately transitions from growth to storage. The latter is dominated by embryos (Brassicaceae and Fabaceae) or endosperms (Gramineae). Intradomain sugar transport occurs symplasmically through plasmodesmata. Interdomain sugar transport relies on plasma-membrane transporters operating in efflux (maternal and endosperm) or influx (endosperm and embryo) modes. Discussed is substantial progress made in identifying, and functionally evaluating, sugar symporters (STPs, SUTs or SUCs) and uniporters (SWEETs). These findings have underpinned a mechanistic understanding of seed loading. Less well researched are possible physical limitations imposed by hydraulic conductivities of differentiating protophloem and of subsequent plasmodesmal transport. The latter is coupled with sugar homeostasis within each domain mediated by sugar transporters. A similar conclusion is ascribed to fragmentary understanding of regulatory mechanisms integrating transport events with seed growth and storage., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Volume and surface area of chloroplasts and surface area of plasmodesmata pit fields are presented for two C 4 species, maize and sugarcane, with respect to cell surface area and cell volume. Serial block face scanning electron microscopy (SBF-SEM) and confocal laser scanning microscopy with the Airyscan system (LSM) were used. Chloroplast size estimates were much faster and easier using LSM than with SBF-SEM; however, the results were more variable than SBF-SEM. Mesophyll cells were lobed where chloroplasts were located, facilitating cell-to-cell connections while allowing for greater intercellular airspace exposure. Bundle sheath cells were cylindrical with chloroplasts arranged centrifugally. Chloroplasts occupied c. 30-50% of mesophyll cell volume, and 60-70% of bundle sheath cell volume. Roughly 2-3% of each cell surface area was covered by plasmodesmata pit fields for both bundle sheath and mesophyll cells. This work will aid future research to develop SBF-SEM methodologies with the aim to better understand the effect of cell structure on C 4 photosynthesis., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Plasmodesmata (PD) facilitate movement of molecules between plant cells. Regulation of this movement is still not understood. Plasmodesmata are hard to study, being deeply embedded within cell walls and incorporating several membrane types. Thus, structure and protein composition of PD remain enigmatic. Previous studies of PD protein composition identified protein lists with few validations, making functional conclusions difficult. We developed a PD scoring approach in iteration with large-scale systematic localization, defining a high-confidence PD proteome of Physcomitrium patens (HC300). HC300, together with bona fide PD proteins from literature, were placed in Pddb. About 65% of proteins in HC300 were not previously PD-localized. Callose-degrading glycolyl hydrolase family 17 (GHL17) is an abundant protein family with representatives across evolutionary scale. Among GHL17s, we exclusively found members of one phylogenetic clade with PD localization and orthologs occur only in species with developed PD. Phylogenetic comparison was expanded to xyloglucan endotransglucosylases/hydrolases and Exordium-like proteins, which also diversified into PD-localized and non-PD-localized members on distinct phylogenetic clades. Our high-confidence PD proteome HC300 provides insights into diversification of large protein families. Iterative and systematic large-scale localization across plant species strengthens the reliability of HC300 as basis for exploring structure, function, and evolution of this important organelle., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

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., (© 2019 The Authors New Phytologist © 2019 New Phytologist Trust.)

Chloroplasts retain part of their ancestral genomes and the machinery for expression of those genomes. The nucleus-encoded chloroplast RNA helicase INCREASED SIZE EXCLUSION LIMIT2 (ISE2) is required for chloroplast ribosomal RNA processing and chloro-ribosome assembly. To further elucidate ISE2's role in chloroplast translation, two independent approaches were used to identify its potential protein partners. Both a yeast two-hybrid screen and a pull-down assay identified plastid ribosomal protein L15, uL15c (formerly RPL15), as interacting with ISE2. The interaction was confirmed in vivo by co-immunoprecipitation. Interestingly, we found that rpl15 null mutants do not complete embryogenesis, indicating that RPL15 is an essential gene for autotrophic growth of Arabidopsis thaliana. Arabidopsis and Nicotiana benthamiana plants with reduced expression of RPL15 developed chlorotic leaves, had reduced photosynthetic capacity and exhibited defective chloroplast development. Processing of chloroplast ribosomal RNAs and assembly of ribosomal subunits were disrupted by reduced expression of RPL15. Chloroplast translation was also decreased, reducing accumulation of chloroplast-encoded proteins, in such plants compared to wild-type plants. Notably, knockdown of RPL15 expression increased intercellular trafficking, a phenotype also observed in plants with reduced ISE2 expression. This finding provides further evidence for chloroplast function in modulating intercellular trafficking via plasmodesmata., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Recent studies have revealed that some responses of fern stomata to environmental signals differ from those of their relatives in seed plants. However, it is unknown whether the biophysical properties of guard cells differ fundamentally between species of both clades. Intracellular micro-electrodes and the fluorescent Ca 2+ reporter FURA2 were used to study voltage-dependent cation channels and Ca 2+ signals in guard cells of the ferns Polypodium vulgare and Asplenium scolopendrium. Voltage clamp experiments with fern guard cells revealed similar properties of voltage-dependent K + channels as found in seed plants. However, fluorescent dyes moved within the fern stomata, from one guard cell to the other, which does not occur in most seed plants. Despite the presence of plasmodesmata, which interconnect fern guard cells, Ca 2+ signals could be elicited in each of the cells individually. Based on the common properties of voltage-dependent channels in ferns and seed plants, it is likely that these key transport proteins are conserved in vascular plants. However, the symplastic connections between fern guard cells in mature stomata indicate that the biophysical mechanisms that control stomatal movements differ between ferns and seed plants., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Contents Summary 62 I. Introduction 62 II. Plasmodesmal regulation is an innate defence response 63 III. Reactive oxygen species regulate plasmodesmal function 63 IV. Plasmodesmal regulation by and of defence-associated small molecules 64 V. Plasmodesmata facilitate systemic defence signalling 64 VI. Virulent pathogens exploit plasmodesmata 66 VII. Outlook 66 Acknowledgements 66 References 66 SUMMARY: 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., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

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 Ca 2+ -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 Ca 2+ -signalling specificity during bacterial pathogen attack and is required for a complete defence response against Pseudomonas syringae., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

The endoplasmic reticulum (ER) is connected to the plasma membrane (PM) through the plant-specific NETWORKED protein, NET3C, and phylogenetically conserved vesicle-associated membrane protein-associated proteins (VAPs). Ten VAP homologues (VAP27-1 to 27-10) can be identified in the Arabidopsis genome and can be divided into three clades. Representative members from each clade were tagged with fluorescent protein and expressed in Nicotiana benthamiana. Proteins from clades I and III localized to the ER as well as to ER/PM contact sites (EPCSs), whereas proteins from clade II were found only at the PM. Some of the VAP27-labelled EPCSs localized to plasmodesmata, and we show that the mobility of VAP27 at EPCSs is influenced by the cell wall. EPCSs closely associate with the cytoskeleton, but their structure is unaffected when the cytoskeleton is removed. VAP27-labelled EPCSs are found in most cell types in Arabidopsis, with the exception of cells in early trichome development. Arabidopsis plants expressing VAP27-GFP fusions exhibit pleiotropic phenotypes, including defects in root hair morphogenesis. A similar effect is also observed in plants expressing VAP27 RNAi. Taken together, these data indicate that VAP27 proteins used at EPCSs are essential for normal ER-cytoskeleton interaction and for plant development., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

The two major vascular conduits in plants, the xylem and phloem, theoretically provide opportunities for the long-distance translocation of almost any type of water-borne molecule. This review focuses on the signalling functions conveyed by the movement of macromolecules. Here, a signal is defined as the communication of information from source to destination, where it modifies development, physiology or defence through altered gene expression or by direct influences on other cellular processes. Xylem and phloem sap both contain diverse classes of proteins; in addition, phloem contains many full-length and small RNA species. Only a few of these mobile molecules have proven functions in signalling. The transduction of signals typically depends on connection to appropriate signalling pathways. Incoming protein signals require specific detection systems, generally via receptors. Mobile RNAs require either the translation or presence of a homologous target. Given that phloem sieve elements are enucleate and lack translation machinery, RNA function requires subsequent unloading at least into adjacent companion cells. The binding of RNA by proteins in ribonucleoprotein complexes enables the translocation of some signals, with evidence for both sequence-specific and size-specific binding. Several examples of long-distance macromolecular signalling are highlighted, including the FT protein signal which regulates flowering time and other developmental switches., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)