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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.)

Background and Aims: Fertilization relies on pollen mother cells able to transition from mitosis to meiosis to supply gametes. This process involves remarkable changes at the molecular, cellular and physiological levels, including (but not limited to) remodelling of the cell wall. During the onset of meiosis, the cellulose content in the pollen mother cell walls gradually declines, with the concurrent deposition of the polysaccharide callose in anther locules. We aim to understand the biological significance of cellulose-to-callose turnover in pollen mother cells walls., Methods: We carried out electron microscopic, aniline blue and renaissance staining analyses of rice flowers., Key Results: Our observations indicate that in wild-type rice anthers, the mitosis-to-meiosis transition coincides with a gradual reduction in the number of cytoplasmic connections called plasmodesmata. A mutant in the Oryza sativa callose synthase GSL5 (Osgsl5-3), impaired in callose accumulation in premeiotic and meiotic anthers, displayed a greater reduction in plasmodesmata frequency among pollen mother cells and tapetal cells, suggesting a role for callose in maintenance of plasmodesmata. In addition, a significant increase in extracellular distance between pollen mother cells and impaired premeiotic cell shaping was observed in the Osgsl5-3 mutant., Conclusions: The results suggest that callose-to-cellulose turnover during the transition from mitosis to meiosis is necessary to maintain cell-to-cell connections and optimal extracellular distance among the central anther locular cells. The findings of this study contribute to our understanding of the regulatory influence of callose metabolism during initiation of meiosis in flowering plants., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved in both plants and animals and play critical roles in activating innate immunity to defend against various pathogens. However, the role of MAPK cascades in positively regulating or enhancing viral infections in plants is unclear. In this study, we investigate the involvement of MAPK cascades in infection by the positive-strand RNA virus tomato chlorosis virus (ToCV). Our findings reveal that ToCV infection activates MAPK cascades, promoting virus spread within plants. Specifically, ToCV P7, a pathogenicity determinant protein, localizes to the plasma membrane and recruits NbMPK3/6 from the nucleus. Subsequently, P7 is directly phosphorylated on serine 59 by NbMPK3/6. Phosphorylated P7 interacts with NbREM1.1 and inhibits its ability to induce callose deposition at plasmodesmata. These results demonstrate that NbMPK3/6 directly phosphorylate ToCV P7, modulating antiviral defence mechanisms by downregulating callose deposition at plasmodesmata and thereby enhancing ToCV transmission in N. benthamiana. This study sheds light on the intricate arms race between host defence and viral counter-defence strategies., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The cytoplasm of most plant cells is connected by membrane-lined cell wall channels, the plasmodesmata (PD). Dynamic regulation of sugar, hormone, and protein diffusion through PD is essential for plant development and stress responses. Understanding this regulation requires knowledge of factors and mechanisms that control PD permeability through the modulation of callose levels in the cell wall around PD openings. We investigated PD regulation in leaf epidermal cells in relation to drought stress in Arabidopsis. PD-mediated cell wall permeability was decreased by drought stress and the hormone abscisic acid (ABA), and we tested how this related to several PD-associated genes with drought-responsive expression. Mutants of NON-RACE SPECIFIC DISEASE RESISTANCE/HIN1 HAIRPIN-INDUCED-LIKE 12 (NHL12) showed relatively low PD permeability that was unaffected by drought or ABA treatment. Overexpression of NHL12 in Nicotiana benthamiana epidermal cells increased PD permeability. Moreover, we showed that NHL12 can potentially interact with the callose synthase regulator NHL3 and we explored the effect of NHL12 abundance and/or lower interface permeability on ABA signaling genes. Our results indicate that NHL12 is a drought-responsive negative regulator of PD callose levels and, thereby, interface permeability. Results are discussed in relation to PD function during drought stress and the regulation of intercellular transport., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Plasmodesmata (PD) are nanochannels that facilitate cell-to-cell transport in plants. More productive and photosynthetically efficient C 4 plants form more PD at the mesophyll (M)-bundle sheath (BS) interface in their leaves than their less efficient C 3 relatives. In C 4 leaves, PD play an essential role in facilitating the rapid metabolite exchange between the M and BS cells to operate a biochemical CO 2 concentrating mechanism, which increases the CO 2 partial pressure at the site of Rubisco in the BS cells and hence photosynthetic efficiency. The genetic mechanism controlling PD formation in C 3 and C 4 leaves is largely unknown, especially in monocot crops, due to the technical challenge of quantifying these nanostructures with electron microscopy. To address this issue, we have generated stably transformed lines of Oryza sativa (rice, C 3 ) and Setaria viridis (setaria, C 4 ) with fluorescent protein-tagged PD to build the first spatiotemporal atlas of leaf pit field (cluster of PD) density in monocots without the need for electron microscopy. Across leaf development, setaria had consistently more PD connections at the M-BS wall interface than rice while the difference in M-M pit field density varied. While light was a critical trigger of PD formation, cell type and function determined leaf pit field density. Complementary temporal mRNA sequencing and gene co-expression network analysis revealed that the pattern of pit field density correlated with differentially expressed PD-associated genes and photosynthesis-related genes. PD-associated genes identified from our co-expression network analysis are related to cell wall expansion, translation and chloroplast signalling., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The control of cell-cell communication via plasmodesmata (PD) plays a key role in plant development. In tree buds, low-temperature conditions (LT) induce a switch in plasmodesmata from a closed to an open state, which restores cell-to-cell communication in the shoot apex and releases dormancy. Using genetic and cell-biological approaches, we have identified a previously uncharacterized transcription factor, Low-temperature-Induced MADS-box 1 (LIM1), as an LT-induced, direct upstream activator of the gibberellic acid (GA) pathway. The LIM1-GA module mediates low temperature-induced plasmodesmata opening, by negatively regulating callose accumulation to promote dormancy release. LIM1 also activates expression of FT1 (FLOWERING LOCUS T), another LT-induced factor, with LIM1-FT1 forming a coherent feedforward loop converging on low-temperature regulation of gibberellin signaling in dormancy release. Mathematical modeling and experimental validation suggest that negative feedback regulation of LIM1 by gibberellin could play a crucial role in maintaining the robust temporal regulation of bud responses to low temperature. These results reveal genetic factors linking temperature control of cell-cell communication with regulation of seasonally-aligned growth crucial for adaptation of trees., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Emerging evidence suggests that the localization of viral movement proteins (MPs) to both plasmodesmata (PD) and viral replication complexes (VRCs) is the key to viral cell-to-cell movement. However, the molecular mechanism that establishes the subcellular localization of MPs is not fully understood. Here, we investigated the PD localization pathway of red clover necrotic mosaic virus (RCNMV) MP and the functional regions of MP that are crucial for MP localization to PD and VRCs. Disruption analysis of the transport pathway suggested that RCNMV MP does not rely on the ER-Golgi pathway or the cytoskeleton for the localization to the PD. Furthermore, mutagenesis analysis identified amino acid residues within the alpha helix regions responsible for localization to the PD or VRCs. These α-helix regions were also essential for efficient viral cell-to-cell movement, highlighting the importance of these dynamic localization of the MPs for viral infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Plasmodesmata are membranous nanopores that connect the cytoplasm of adjacent plant cells and enable the cell-to-cell trafficking of nutrients, macromolecules, as well as invading viruses. Plasmodesmata play fundamental roles in the regulation of intercellular communication, contributing to plant development, environmental responses, and interactions with viral pathogens. Discovering plasmodesmal localization of plant or viral proteins could provide useful functional information about the protein and is important for understanding the mechanisms of plant-virus interactions. To facilitate these studies, we describe a protocol for confocal microscopy-based analysis of different plasmodesmal targeting proteins to select the best plasmodesmal marker for studying the virus-plasmodesmata interactions or plasmodesmal transport. Specifically, the analyses of these events are illustrated using the cell-to-cell movement protein (MP) of the Turnip vein-clearing virus (TVCV), the Arabidopsis Plasmodesmata-Localized Protein 5 (PDLP5) and Plasmodesmata Callose-Binding Protein 1 (PDCB1). The protein plasmodesmal localization data are analyzed in parallel with the global visualization of plasmodesmata using aniline blue staining of the sampled tissues. These approaches can be easily adapted to analyze the plasmodesmal localization of any cellular or pathogen proteins in planta.

Diverging from conventional cell division models, plant cells undergo incomplete division to generate plasmodesmata communication bridges between daughter cells. Although fundamental for plant multicellularity, the molecular events leading to bridge stabilization, as opposed to severing, remain unknown. Using electron tomography, we mapped the transition from cell plate fenestrae to plasmodesmata. We show that the endoplasmic reticulum (ER) connects daughter cells across fenestrae, and as the cell plate matures, fenestrae contract, causing the plasma membrane (PM) to mold around constricted ER tubes. The ER's presence prevents fenestrae fusion, forming plasmodesmata, whereas its absence results in closure. The ER-PM protein tethers MCTP3, MCTP4, and MCTP6 further stabilize nascent plasmodesmata during fenestrae contraction. Genetic deletion in Arabidopsis reduces plasmodesmata formation. Our findings reveal how plants undergo incomplete division to promote intercellular communication.

The microdomains of plasmodesmata, specialized cell-wall channels responsible for communications between neighboring cells, are composed of various plasmodesmata-located proteins (PDLPs) and lipids. Here, we found that, among all PDLP or homologous proteins in Arabidopsis thaliana genome, PDLP5 and PDLP7 possessed a C-terminal sphingolipid-binding motif, with the latter being the only member that was significantly upregulated upon turnip mosaic virus and cucumber mosaic virus infections. pdlp7 mutant plants exhibited significantly reduced callose deposition, larger plasmodesmata diameters, and faster viral transmission. These plants exhibited increased glucosidase activity but no change in callose synthase activity. PDLP7 interacted specifically with glucan endo-1,3-β-glucosidase 10 (BG10). Consistently, higher levels of callose deposition and slower virus transmission in bg10 mutants were observed. The interaction between PDLP7 and BG10 was found to depend on the presence of the Gnk2-homologous 1 (GnK2-1) domain at the N terminus of PDLP7 with Asp-35, Cys-42, Gln-44, and Leu-116 being essential. In vitro supplementation of callose was able to change the conformation of the GnK2-1 domain. Our data suggest that the GnK2-1 domain of PDLP7, in conjunction with callose and BG10, plays a key role in plasmodesmata opening and closure, which is necessary for intercellular movement of various molecules., (Copyright © 2024 Science China Press. Published by Elsevier B.V. All rights reserved.)

Pear chlorotic leaf spot-associated virus (PCLSaV) is a newly described emaravirus that infects pear trees. The virus genome consists of at least five single-stranded, negative-sense RNAs. The P5 encoded by RNA5 is unique to PCLSaV. In this study, the RNA silencing suppression (RSS) activity of P5 and its subcellular localization were determined in Nicotiana benthamiana plants by Agrobacterium tumefaciens-mediated expression assays and green fluorescent protein RNA silencing induction. Protein P5 partially suppressed local RNA silencing, strongly suppressed systemic RNA silencing and triggered reactive oxygen species accumulation. The P5 self-interacted and showed subcellular locations in plasmodesmata, endoplasmic reticulum and nucleus. Furthermore, P5 rescued the cell-to-cell movement of a movement defective mutant PVXΔP25 of potato virus X (PVX) and enhanced the pathogenicity of PVX. The N-terminal 1-89 amino acids of the P5 were responsible for the self-interaction ability and RSS activity, for which the signal peptide at positions 1-19 was indispensable. This study demonstrated the function of an emaravirus protein as a pathogenic factor suppressing plant RNA silencing to enhance virus infection and as an enhancer of virus movement., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

FW2.2 (standing for FRUIT WEIGHT 2.2), the founding member of the CELL NUMBER REGULATOR (CNR) gene family, was the first cloned gene underlying a quantitative trait locus (QTL) governing fruit size and weight in tomato (Solanum lycopersicum). However, despite this discovery over 20 yr ago, the molecular mechanisms by which FW2.2 negatively regulates cell division during fruit growth remain undeciphered. In the present study, we confirmed that FW2.2 is a membrane-anchored protein whose N- and C-terminal ends face the apoplast. We unexpectedly found that FW2.2 is located at plasmodesmata (PD). FW2.2 participates in the spatiotemporal regulation of callose deposition at PD and belongs to a protein complex which encompasses callose synthases. These results suggest that FW2.2 has a regulatory role in cell-to-cell communication by modulating PD transport capacity and trafficking of signaling molecules during fruit development., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Plasmodesmata (PDs) are intercellular organelles carrying multiple membranous nanochannels that allow the trafficking of cellular signalling molecules. The channel regulation of PDs occurs dynamically and is required in various developmental and physiological processes. It is well known that callose is a critical component in regulating PD permeability or symplasmic connectivity, but the understanding of the signalling pathways and mechanisms of its regulation is limited. Here, we used the reverse genetic approach to investigate the role of C-type lectin receptor-like kinase 1 (CLRLK1) in the aspect of PD callose-modulated symplasmic continuity. Here, we found that loss-of-function mutations in CLRLK1 resulted in excessive PD callose deposits and reduced symplasmic continuity, resulting in an accelerated gravitropic response. The protein interactome study also found that CLRLK1 interacted with actin depolymerizing factor 3 (ADF3) in vitro and in plants. Moreover, mutations in ADF3 result in elevated PD callose deposits and faster gravitropic response. Our results indicate that CLRLK1 and ADF3 negatively regulate PD callose accumulation, contributing to fine-tuning symplasmic opening apertures. Overall, our studies identified two key components involved in the deposits of PD callose and provided new insights into how symplasmic connectivity is maintained by the control of PD callose homoeostasis., (© 2024 The Author(s). Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Competing Interests: Conflict of interest statement. The authors do not declare any conflict of interest.

Cassava (Manihot esculenta) is a deciduous woody perennial shrub that stores large amounts of carbon and water in its storage roots. Previous studies have shown that assimilating unloading into storage roots happens symplasmically once secondary anatomy is established. However, mechanisms controlling phloem loading and overall carbon partitioning to different cassava tissues remain unclear. Here, we used a combination of histological, transcriptional, and biochemical analyses on different cassava tissues and at different timepoints to better understand source-sink carbon allocation. We found that cassava likely utilizes a predominantly passive symplasmic phloem loading strategy, indicated by the lack of expression of genes coding for key players of sucrose transport, the existence of branched plasmodesmata in the companion cell/bundle sheath interface of minor leaf veins, and very high leaf sucrose concentrations. Furthermore, we showed that tissue-specific changes in anatomy and non-structural carbohydrate contents are associated with tissue-specific modification in gene expression for sucrose cleavage/synthesis, as well as subcellular compartmentalization of sugars. Overall, our data suggest that carbon allocation during storage root filling is mostly facilitated symplasmically and is likely mostly regulated by local tissue demand and subcellular compartmentalization., Competing Interests: Conflict of interest statement. The authors have declared that no competing interest exists., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Intercellular communication in plants, as in other multicellular organisms, allows cells in tissues to coordinate their responses for development and in response to environmental stimuli. Much of this communication is facilitated by plasmodesmata (PD), consisting of membranes and cytoplasm, that connect adjacent cells to each other. PD have long been viewed as passive conduits for the movement of a variety of metabolites and molecular cargoes, but this perception has been changing over the last two decades or so. Research from the last few years has revealed the importance of PD as signaling hubs and as crucial players in hormone signaling. The adoption of advanced biochemical approaches, molecular tools, and high-resolution imaging modalities has led to several recent breakthroughs in our understanding of the roles of PD, revealing the structural and regulatory complexity of these 'protoplasmic connecting threads'. We highlight several of these findings that we think well illustrate the current understanding of PD as functioning at the nexus of plant physiology, development, and acclimation to the environment., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Plasmodesmata connect adjoining plant cells, allowing molecules to move between the connected cells for communication and sharing resources. It has been well established that the plant polysaccharide callose is deposited at plasmodesmata, regulating their aperture and function. Among proteins involved in maintaining callose homeostasis, PLASMODESMATA-LOCATED PROTEINSs (PDLPs) promote callose deposition at plasmodesmata. This study explored the function of PDLP5 and PDLP6 in different cell types. We discovered that PDLP5 and PDLP6 are expressed in nonoverlapping cell types in Arabidopsis (Arabidopsis thaliana). The overexpression of PDLP5 and PDLP6 results in the overaccumulation of plasmodesmal callose at different cell interfaces, indicating that PDLP5 and PDLP6 are active in different cell types. We also observed 2 distinct patterns of starch accumulation in mature leaves of PDLP5 and PDLP6 overexpressors. An enzyme-catalyzed proximity labeling approach was used to identify putative functional partners of the PDLPs. We identified SUCROSE SYNTHASE 6 (SUS6) as a functional partner of PDLP6 in the vasculature. We further demonstrated that PDLP6 physically and genetically interacts with SUS6. In addition, CALLOSE SYNTHASE 7 (CALS7) physically interacts with SUS6 and PDLP6. Genetic interaction studies showed that CALS7 is required for PDLP6 function. We propose that PDLP6 functions with SUS6 and CALS7 in the vasculature to regulate plasmodesmal function., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Reactive oxygen species (ROS) are important signalling molecules that influence many aspects of plant biology. One way in which ROS influence plant growth and development is by modifying intercellular trafficking through plasmodesmata (PD). Viruses have evolved to use PD for their local cell-to-cell spread between plant cells, so it is therefore not surprising that they have found ways to modulate ROS and redox signalling to optimise PD function for their benefit. This review examines how intracellular signalling via ROS and redox pathways regulate intercellular trafficking via PD during development and stress. The relationship between viruses and ROS-redox systems, and the strategies viruses employ to control PD function by interfering with ROS-redox in plants is also discussed., (© 2024 John Wiley & Sons Ltd.)

Callose, a β-1,3-glucan plant cell wall polymer, regulates symplasmic channel size at plasmodesmata (PD) and plays a crucial role in a variety of plant processes. However, elucidating the molecular mechanism of PD callose homeostasis is limited. We screened and identified an Arabidopsis mutant plant with excessive callose deposition at PD and found that the mutated gene was α1-COP, a member of the coat protein I (COPI) coatomer complex. We report that loss of function of α1-COP elevates the callose accumulation at PD by affecting subcellular protein localization of callose degradation enzyme PdBG2. This process is linked to the functions of ERH1, an inositol phosphoryl ceramide synthase, and glucosylceramide synthase through physical interactions with the α1-COP protein. Additionally, the loss of function of α1-COP alters the subcellular localization of ERH1 and GCS proteins, resulting in a reduction of GlcCers and GlcHCers molecules, which are key sphingolipid (SL) species for lipid raft formation. Our findings suggest that α1-COP protein, together with SL modifiers controlling lipid raft compositions, regulates the subcellular localization of GPI-anchored PDBG2 proteins, and hence the callose turnover at PD and symplasmic movement of biomolecules. Our findings provide the first key clue to link the COPI-mediated intracellular trafficking pathway to the callose-mediated intercellular signaling pathway through PD., (© 2024 The Authors. Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

Competing Interests: Conflict of interest statement. None declared.

Correct regulation of intercellular communication is a fundamental requirement for cell differentiation. In Arabidopsis thaliana, the female germline differentiates from a single somatic ovule cell that becomes encased in β-1,3-glucan, a water insoluble polysaccharide implicated in limiting pathogen invasion, regulating intercellular trafficking in roots, and promoting pollen development. Whether β-1,3-glucan facilitates germline isolation and development has remained contentious, since limited evidence is available to support a functional role. Here, transcriptional profiling of adjoining germline and somatic cells revealed differences in gene expression related to β-1,3-glucan metabolism and signalling through intercellular channels (plasmodesmata). Dominant expression of a β-1,3-glucanase in the female germline transiently perturbed β-1,3-glucan deposits, allowed intercellular movement of tracer molecules, and led to changes in germline gene expression and histone marks, eventually leading to termination of germline development. Our findings indicate that germline β-1,3-glucan fulfils a functional role in the ovule by insulating the primary germline cell, and thereby determines the success of downstream female gametogenesis., (© 2024. The Author(s).)

Main Conclusion: Developing bryophytes differentially modify their plasmodesmata structure and function. Secondary plasmodesmata formation via twinning appears to be an ancestral trait. Plasmodesmata networks in hornwort sporophyte meristems resemble those of angiosperms. All land-plant taxa use plasmodesmata (PD) cell connections for symplasmic communication. In angiosperm development, PD networks undergo an extensive remodeling by structural and functional PD modifications, and by postcytokinetic formation of additional secondary PD (secPD). Since comparable information on PD dynamics is scarce for the embryophyte sister groups, we investigated maturating tissues of Anthoceros agrestis (hornwort), Physcomitrium patens (moss), and Marchantia polymorpha (liverwort). As in angiosperms, quantitative electron microscopy revealed secPD formation via twinning in gametophytes of all model bryophytes, which gives rise to laterally adjacent PD pairs or to complex branched PD. This finding suggests that PD twinning is an ancient evolutionary mechanism to adjust PD numbers during wall expansion. Moreover, all bryophyte gametophytes modify their existing PD via taxon-specific strategies resembling those of angiosperms. Development of type II-like PD morphotypes with enlarged diameters or formation of pit pairs might be required to maintain PD transport rates during wall thickening. Similar to angiosperm leaves, fluorescence redistribution after photobleaching revealed a considerable reduction of the PD permeability in maturating P. patens phyllids. In contrast to previous reports on monoplex meristems of bryophyte gametophytes with single initials, we observed targeted secPD formation in the multi-initial basal meristems of A. agrestis sporophytes. Their PD networks share typical features of multi-initial angiosperm meristems, which may hint at a putative homologous origin. We also discuss that monoplex and multi-initial meristems may require distinct types of PD networks, with or without secPD formation, to control maintenance of initial identity and positional signaling., (© 2024. The Author(s).)

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.)

Multicellularity has emerged multiple times in evolution, enabling groups of cells to share a living space and reducing the burden of solitary tasks. While unicellular organisms exhibit individuality and independence, cooperation among cells in multicellular organisms brings specialization and flexibility. However, multicellularity also necessitates intercellular dependence and relies on intercellular communication. In plants, this communication is facilitated by plasmodesmata: intercellular bridges that allow the direct (cytoplasm-to-cytoplasm) transfer of information between cells. Plasmodesmata transport essential molecules that regulate plant growth, development, and stress responses. They are embedded in the extracellular matrix but exhibit flexibility, adapting intercellular flux to meet the plant's needs.In this review, we delve into the formation and functionality of plasmodesmata and examine the capacity of the plant communication network to respond to developmental and environmental cues. We illustrate how environmental pressure shapes cellular interactions and aids the plant in adapting its growth.

Plasmodesmata are transmembrane channels embedded within the cell wall that can facilitate the intercellular communication in plants. Plasmodesmata callose-binding (PDCB) protein that associates with the plasmodesmata contributes to cell wall extension. Given that the elongation of cotton fiber cells correlates with the dynamics of the cell wall, this protein can be related to the cotton fiber elongation. This study sought to identify PDCB family members within the Gossypium. hirsutum genome and to elucidate their expression profiles. A total of 45 distinct family members were observed through the identification and screening processes. The analysis of their physicochemical properties revealed the similarity in the amino acid composition and molecular weight across most members. The phylogenetic analysis facilitated the construction of an evolutionary tree, categorizing these members into five groups mainly distributed on 20 chromosomes. The fine mapping results facilitated a tissue-specific examination of group V, revealing that the expression level of GhPDCB9 peaked five days after flowering. The VIGS experiments resulted in a marked decrease in the gene expression level and a significant reduction in the mature fiber length, averaging a shortening of 1.43-4.77 mm. The results indicated that GhPDCB9 played a pivotal role in the cotton fiber development and served as a candidate for enhancing cotton yield., Competing Interests: The authors declare there are no competing interests., (©2024 Zhang et al.)

Viruses employ a series of diverse translational strategies to expand their coding capacity, which produces viral proteins with common domains and entangles virus-host interactions. P3N-PIPO, which is a transcriptional slippage product from the P3 cistron, is a potyviral protein dedicated to intercellular movement. Here, we show that P3N-PIPO from watermelon mosaic virus (WMV) triggers cell death when transiently expressed in Cucumis melo accession PI 414723 carrying the Wmr resistance gene. Surprisingly, expression of the P3N domain, shared by both P3N-PIPO and P3, can alone induce cell death, whereas expression of P3 fails to activate cell death in PI 414723. Confocal microscopy analysis revealed that P3N-PIPO targets plasmodesmata (PD) and P3N associates with PD, while P3 localizes in endoplasmic reticulum in melon cells. We also found that mutations in residues L35, L38, P41, and I43 of the P3N domain individually disrupt the cell death induced by P3N-PIPO, but do not affect the PD localization of P3N-PIPO. Furthermore, WMV mutants with L35A or I43A can systemically infect PI 414723 plants. These key residues guide us to discover some WMV isolates potentially breaking the Wmr resistance. Through searching the NCBI database, we discovered some WMV isolates with variations in these key sites, and one naturally occurring I43V variation enables WMV to systemically infect PI 414723 plants. Taken together, these results demonstrate that P3N-PIPO, but not P3, is the avirulence determinant recognized by Wmr, although the shared N terminal P3N domain can alone trigger cell death.IMPORTANCEThis work reveals a novel viral avirulence (Avr) gene recognized by a resistance (R) gene. This novel viral Avr gene is special because it is a transcriptional slippage product from another virus gene, which means that their encoding proteins share the common N-terminal domain but have distinct C-terminal domains. Amazingly, we found that it is the common N-terminal domain that determines the Avr-R recognition, but only one of the viral proteins can be recognized by the R protein to induce cell death. Next, we found that these two viral proteins target different subcellular compartments. In addition, we discovered some virus isolates with variations in the common N-terminal domain and one naturally occurring variation that enables the virus to overcome the resistance. These results show how viral proteins with common domains interact with a host resistance protein and provide new evidence for the arms race between plants and viruses., Competing Interests: The authors declare no conflict of interest.

Fusarium head blight disease on small-grain cereals is primarily caused by the ascomycete fungal pathogen Fusarium graminearum. Infection of floral spike tissues is characterized by the biosynthesis and secretion of potent trichothecene mycotoxins, of which deoxynivalenol (DON) is widely reported due to its negative impacts on grain quality and consumer safety. The TRI5 gene encodes an essential enzyme in the DON biosynthesis pathway and the single gene deletion mutant, ΔTri5, is widely reported to restrict disease progression to the inoculated spikelet. In this study, we present novel bioimaging evidence revealing that DON facilitates the traversal of the cell wall through plasmodesmata, a process essential for successful colonization of host tissue. Chemical complementation of ΔTri5 did not restore macro- or microscopic phenotypes, indicating that DON secretion is tightly regulated both spatially and temporally. A comparative qualitative and quantitative morphological cellular analysis revealed infections had no impact on plant cell wall thickness. Immunolabelling of callose at plasmodesmata during infection indicates that DON can increase deposits when applied exogenously but is reduced when F. graminearum hyphae are present. This study highlights the complexity of the interconnected roles of mycotoxin production, cell wall architecture and plasmodesmata in this highly specialized interaction., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Messenger RNAs (mRNAs) are the templates for protein translation but can also act as non-cell-autonomous signaling molecules. Plants input endogenous and exogenous cues to mobile mRNAs and output them to local or systemic target cells and organs to support specific plant responses. Mobile mRNAs form ribonucleoprotein (RNP) complexes with proteins during transport. Components of these RNP complexes could interact with plasmodesmata (PDs), a major mediator of mRNA transport, to ensure mRNA mobility and transport selectivity. Based on advances in the last two to three years, this review summarizes mRNA transport mechanisms in local and systemic signaling from the perspective of RNP complex formation and PD transport. We also discuss the physiological roles of endogenous mRNA transport and the recently revealed roles of non-cell-autonomous mRNAs in inter-organism communication., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Callose, a β-(1,3)-d-glucan polymer, is essential for regulating intercellular trafficking via plasmodesmata (PD). Pathogens manipulate PD-localized proteins to enable intercellular trafficking by removing callose at PD or, conversely, by increasing callose accumulation at PD to limit intercellular trafficking during infection. Plant defense hormones like salicylic acid regulate PD-localized proteins to control PD and intercellular trafficking during immune defense responses such as systemic acquired resistance. Measuring callose deposition at PD in plants has therefore emerged as a popular parameter for assessing likely intercellular trafficking activity during plant immunity. Despite the popularity of this metric, there is no standard for how these measurements should be made. In this study, three commonly used methods for identifying and quantifying plasmodesmal callose by aniline blue staining were evaluated to determine the most effective in the Nicotiana benthamiana leaf model. The results reveal that the most reliable method used aniline blue staining and fluorescence microscopy to measure callose deposition in fixed tissue. Manual or semiautomated workflows for image analysis were also compared and found to produce similar results, although the semiautomated workflow produced a wider distribution of data points. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Viral diseases seriously threaten rice production. Plasmodesmata (PD)-associated proteins are deemed to play a key role in viral infection in host plants. However, few PD-associated proteins have been discovered in rice to afford viral infection. Here, inspired by the infection mechanism in insect vectors, we identified a member of the Flotillin family taking part in the cell-to-cell transport of rice stripe virus (RSV) in rice. Flotillin1 interacted with RSV nucleocapsid protein (NP) and was localized on PD. In flotillin1 knockout mutant rice, which displayed normal growth, RSV intercellular movement was retarded, leading to significantly decreased disease incidence. The PD pore sizes of the mutant rice were smaller than those of the wild type due to more callose deposits, which was closely related to the upregulation of two callose synthase genes. RSV infection stimulated flotillin1 expression and enlarged the PD aperture via RSV NP. In addition, flotillin1 knockout decreased disease incidences of southern rice black-streaked dwarf virus (SRBSDV) and rice dwarf virus (RDV) in rice. Overall, our study reveals a new PD-associated protein facilitating virus cell-to-cell trafficking and presents the potential of flotillin1 as a target to produce broad-spectrum antiviral rice resources in the future., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

It has been discovered that plant pathogens produce effectors that spread via plasmodesmata (PD) to allow modulation of host processes in distal uninfected cells. Fusarium oxysporum f. sp. lycopersici ( Fol ) facilitates effector translocation by expansion of the size-exclusion limit of PD using the Six5/Avr2 effector pair. How other fungal pathogens manipulate PD is unknown. We recently reported that many fungal pathogens belonging to different families carry effector pairs that resemble the SIX5 / AVR2 gene pair from Fol. Here, we performed structural predictions of three of these effector pairs from Leptosphaeria maculans ( Lm ) and tested their ability to manipulate PD and to complement the virulence defect of a Fol SIX5 knockout mutant. We show that the AvrLm10A homologs are structurally related to FolSix5 and localize at PD when they are expressed with their paired effectors. Furthermore, these effectors were found to complement Fol Six5 function in cell-to-cell mobility assays and in fungal virulence. We conclude that distantly related fungal species rely on structurally related paired effector proteins to manipulate PD and facilitate effector mobility. The wide distribution of these effector pairs implies Six5-mediated effector translocation to be a conserved propensity among fungal plant pathogens. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

In plants, plasmodesmata establish cytoplasmic continuity between cells to allow for communication and resource exchange across the cell wall. While plant pathogens use plasmodesmata as a pathway for both molecular and physical invasion, the benefits of molecular invasion (cell-to-cell movement of pathogen effectors) are poorly understood. To establish a methodology for identification and characterization of the cell-to-cell mobility of effectors, we performed a quantitative live imaging-based screen of candidate effectors of the fungal pathogen Colletotrichum higginsianum . We predicted C. higginsianum effectors by their expression profiles, the presence of a secretion signal, and their predicted and in planta localization when fused to green fluorescent protein. We assayed for cell-to-cell mobility of nucleocytosolic effectors and identified 14 that are cell-to-cell mobile. We identified that three of these effectors are "hypermobile," showing cell-to-cell mobility greater than expected for a protein of that size. To explore the mechanism of hypermobility, we chose two hypermobile effectors and measured their impact on plasmodesmata function and found that even though they show no direct association with plasmodesmata, each increases the transport capacity of plasmodesmata. Thus, our methods for quantitative analysis of cell-to-cell mobility of candidate microbe-derived effectors, or any suite of host proteins, can identify cell-to-cell hypermobility and offer greater understanding of how proteins affect plasmodesmal function and intercellular connectivity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Plasmodesmata are conduits in plant cell walls that allow neighboring cells to communicate and exchange resources. Despite their central importance to plant development and physiology, our understanding of plasmodesmata is relatively limited compared to other subcellular structures. In recent years, technical advances in electron microscopy, mass spectrometry, and phylogenomics have illuminated the structure, composition, and evolution of plasmodesmata in diverse plant lineages. In parallel, forward genetic screens have revealed key signaling pathways that converge to regulate plasmodesmatal transport, including chloroplast-derived retrograde signaling, phytohormone signaling, and metabolic regulation by the conserved eukaryotic Target of Rapamycin kinase. This review summarizes our current knowledge of the structure, evolution, and regulation of plasmodesmatal transport in plants., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

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.)

Plants convert external cues into mobile mRNAs to synchronize meristematic differentiation with environmental dynamics. These mRNAs are selectively transported to intercellular pores, plasmodesmata (PD), for cell-to-cell movement. However, how plants recognize and deliver mobile mRNAs to PD remains unknown. Here we show that mobile mRNAs hitchhike on organelle trafficking to transport towards PD. Perturbed cytoskeleton organization or organelle trafficking severely disrupts the subcellular distribution of mobile mRNAs. Arabidopsis rotamase cyclophilins (ROCs), which are organelle-localized RNA-binding proteins, specifically bind mobile mRNAs on the surface of organelles to direct intracellular transport. Arabidopsis roc mutants exhibit phenotype alterations and disruptions in the transport of mobile mRNAs. These findings suggest that ROCs play a crucial role in facilitating the systemic delivery of mobile mRNAs. Our results highlight that an RNA-binding protein-mediated hitchhiking system is specifically recruited to orient plant mobile mRNAs for intercellular transport., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Cell-to-cell communication via membranous channels called plasmodesmata (PD) plays critical roles during plant development and in response to biotic and abiotic stresses. Several enzymes and receptor-like proteins (RLPs), including Arabidopsis thaliana glucan synthase-likes (GSLs), also known as callose synthases (CALSs), and PD-located proteins (PDLPs), have been implicated in plasmodesmal permeability regulation and intercellular communication. Localization of PDLPs to punctate structures at the cell periphery and their receptor-like identity have raised the hypothesis that PDLPs are involved in the regulation of symplastic trafficking during plant development and in response to endogenous and exogenous signals. Indeed, it was shown that PDLP5 could limit plasmodesmal permeability through inducing an increase in callose accumulation at PD. However, mechanistically, how this is achieved remains to be elucidated. To address this key issue in understanding the regulation of PD, physical and functional interactions between PDLPs and GSLs (using the PDLP5-GSL8/CALS10 pair as a model) were investigated. Our results show that GSL8/CALS10 plays essential roles and is required for the function and plasmodesmal localization of PDLP5. Furthermore, it was demonstrated that the localization of PDLP5 to PD and its function in inducing callose deposition are GSL8-dependent. Importantly, our transgenic study shows that three key members of the GSL family, i.e., GSL5/CALS12, GSL8/CALS10, and GSL12/CALS3, localize to PD and co-localize with PDLP5, suggesting that GSL8/CALS10 might not be the only callose synthase with the determining role in PD regulation. These findings, together with our previous observation showing the direct interaction of GSL8/CALS10 with PDLP5, indicate the pivotal role of the GSL8/CALS10-PDLP5 interplay in regulating PD permeability. Future work is needed to investigate whether the PDLP5 functionality and localization are also disrupted in gsl5 and gsl12 , or it is just gsl8 -specific.

Stone cell, a type of lignified cell, is a unique trait in pear and one of the key factors affects pear fruit quality and economic value. The transmissibility of cell lignification process has been proven to exist, however the effects of callose on the permeability of plasmodesmata (PD) and how to influence cell lignification processes are still unknown. In this study, the genome-wide analysis of PD callose binding proteins (PDCB) gene family in pear genome was performed, and 25 PbPDCB genes were identified and divided into four branches. Similar intron/exon structural patterns were observed in the same branch, strongly supporting their close evolutionary relationship. The expression of PbPDCB16 was negatively correlated with lignin accumulation through qRT-PCR analysis. With transient expression in pear fruit and stable expression in pear calli, the increased callose content accompanied by decreased lignin content was further observed. Besides, compared with wild type Arabidopsis, the transgenic plants grew slowly, and cell walls in the stem were thinner, while fewer PDs were observed on the cell walls, and the interspore filaments were also blocked in transgenic Arabidopsis through the transmission electron microscope (TEM). In summary, overexpression of PbPDCB16 could promote accumulation of callose at PD to affect the PD-mediated intercellular connectivity, and inhibit the intercellular communication. This study will provide new insight in reducing the lignin content through callose deposition, and also provide the theoretical basis for further exploration of lignin metabolism and cell wall lignification to form stone cells in pear fruit., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Plants are exposed to a variety of biotic and abiotic stresses, including wounding at the stem. The healing process (tissue reunion) begins immediately after stem wounding. The plant hormone auxin plays an important role during tissue reunion. In decapitated stems, auxin transport from the shoot apex is reduced and tissue reunion does not occur but is restored by application of indole-3-acetic acid (IAA). In this study, we found that plasmodesmata callose binding protein 2 (PDCB2) affects the expansion of the cambium/phloem region via changes in auxin response during the process of tissue reunion. PDCB2 was expressed in the cortex and endodermis on the incised side of stems 1-3 days after incision. PDCB2-knockout plants showed reduced callose deposition at plasmodesmata and DR5::GUS activity in the endodermis/cortex in the upper region of the incision accompanied by an increase in size of the cambium/phloem region during tissue reunion. In addition, PIN(PIN-FORMED)3, which is involved in lateral auxin transport, was induced by auxin in the cambium/phloem and endodermis/cortex in the upper part of the incision in wild type, but its expression of PIN3 was decreased in pdcb2 mutant. Our results suggest that PDCB2 contributes to the regulation of cambium/phloem development via auxin response., (© 2023. The Author(s) under exclusive licence to The Botanical Society of Japan.)

Brassinosteroids (BRs) are steroidal phytohormones that are essential for plant growth, development and adaptation to environmental stresses. BRs act in a dose-dependent manner and do not travel over long distances; hence, BR homeostasis maintenance is critical for their function. Biosynthesis of bioactive BRs relies on the cell-to-cell movement of hormone precursors. However, the mechanism of the short-distance BR transport is unknown, and its contribution to the control of endogenous BR levels remains unexplored. Here we demonstrate that plasmodesmata (PD) mediate the passage of BRs between neighboring cells. Intracellular BR content, in turn, is capable of modulating PD permeability to optimize its own mobility, thereby manipulating BR biosynthesis and signaling. Our work uncovers a thus far unknown mode of steroid transport in eukaryotes and exposes an additional layer of BR homeostasis regulation in plants., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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.)

Plasmodesmata (PD) are plasma membrane-lined cytoplasmic nanochannels that mediate cell-to-cell communication across the cell wall. A range of proteins are embedded in the PD plasma membrane and endoplasmic reticulum (ER), and function in regulating PD-mediated symplasmic trafficking. However, knowledge of the nature and function of the ER-embedded proteins in the intercellular movement of non-cell-autonomous proteins is limited. Here, we report the functional characterization of two ER luminal proteins, AtBiP1/2, and two ER integral membrane proteins, AtERdj2A/B, which are located within the PD. These PD proteins were identified as interacting proteins with cucumber mosaic virus (CMV) movement protein (MP) in co-immunoprecipitation studies using an Arabidopsis-derived plasmodesmal-enriched cell wall protein preparation (PECP). The AtBiP1/2 PD location was confirmed by TEM-based immunolocalization, and their AtBiP1/2 signal peptides (SPs) function in PD targeting. In vitro/in vivo pull-down assays revealed the association between AtBiP1/2 and CMV MP, mediated by AtERdj2A, through the formation of an AtBiP1/2-AtERdj2-CMV MP complex within PD. The role of this complex in CMV infection was established, as systemic infection was retarded in bip1/bip2w and erdj2b mutants. Our findings provide a model for a mechanism by which the CMV MP mediates cell-to-cell trafficking of its viral ribonucleoprotein complex., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Effective cellular signaling relies on precise spatial localization and dynamic interactions among proteins in specific subcellular compartments or niches, such as cell-to-cell contact sites and junctions. In plants, endogenous and pathogenic proteins gained the ability to target plasmodesmata, membrane-lined cytoplasmic connections, through evolution to regulate or exploit cellular signaling across cell wall boundaries. For example, the receptor-like membrane protein PLASMODESMATA-LOCATED PROTEIN 5 (PDLP5), a potent regulator of plasmodesmal permeability, generates feed-forward or feed-back signals important for plant immunity and root development. However, the molecular features that determine the plasmodesmal association of PDLP5 or other proteins remain largely unknown, and no protein motifs have been identified as plasmodesmal targeting signals. Here, we developed an approach combining custom-built machine-learning algorithms and targeted mutagenesis to examine PDLP5 in Arabidopsis thaliana and Nicotiana benthamiana. We report that PDLP5 and its closely related proteins carry unconventional targeting signals consisting of short stretches of amino acids. PDLP5 contains 2 divergent, tandemly arranged signals, either of which is sufficient for localization and biological function in regulating viral movement through plasmodesmata. Notably, plasmodesmal targeting signals exhibit little sequence conservation but are located similarly proximal to the membrane. These features appear to be a common theme in plasmodesmal targeting., Competing Interests: Conflict of interest statement. The authors declare that they have no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

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.)

In the present study, we present callus grafting, comprising a method for reproducibly generating tissue chimeras from callus cultures of Arabidopsis thaliana. In this way, callus cultures of different genetic backgrounds may be co-cultivated such that cell-to-cell connectivity is achieved as a chimeric tissue is formed. To track intercellular connectivity and transport between non-clonal callus cells, we used transgenic lines expressing fluorescently tagged mobile and non-mobile fusion constructs. Using fluorescently-labelled reporter lines that label plasmodesmata, we show that secondary complex plasmodesmata are present at the cell walls of connected cells. We use this system to investigate cell-to-cell transport across the callus graft junction and show that different proteins and RNAs are mobile between non-clonal callus cells. Finally, we take advantage of the callus culture system to probe intercellular connectivity of grafted leaf and root calli and the effect of different light regimes of cell-to-cell transport. Taking advantage of the ability of callus to be cultivated in the complete absence of light, we show that the rate of silencing spread is significantly decreased in chimeric calli cultivated in total darkness. We propose that callus grafting is a fast and reliable method for analysing the capacity of a macromolecule to be exchanged between cells independent of the vasculature., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Grafting is widely used to improve the stress tolerance and the fruit yield of horticultural crops. Ribonucleoprotein complexes formed by mRNAs and proteins play critical roles in the communication between scions and stocks of grafted plants. In Pyrus betulaefolia, ankyrin was identified previously to promote the long-distance movement of the ribonucleoprotein complex( PbWoxT1- PbPTB3) by facilitating callose degradation at plasmodesmata. However, the mechanism of the ankyrin-mediated callose degradation remains elusive. In this study, we discovered a β-1,3-glucanase (EC 3.2.1.39, PbPDBG) using ankyrin as a bait from plasmodesmata by co-immunoprecipitation and mass spectrometry. Ankyrin was required for the plasmodesmata-localization of PbPDBG. The grafting and bombardment experiments indicated that overexpressing PbPDBG resulted in decreased callose content at plasmodesmata, and thereby promoting the long-distance transport of the ribonucleoprotein complex. Altogether, our findings revealed that PbPDBG was the key factor in ankyrin-mediated callose degradation at plasmodesmata.

The plant immune system relies on the perception of molecules that signal the presence of a microbe threat. This triggers signal transduction that mediates a range of cellular responses via a collection of molecular machinery including receptors, small molecules, and enzymes. One response to pathogen perception is the restriction of cell-to-cell communication by plasmodesmal closure. We previously found that while chitin and flg22 trigger specialized immune signaling cascades in the plasmodesmal plasma membrane, both execute plasmodesmal closure via callose synthesis at the plasmodesmata. Therefore, the signaling pathways ultimately converge at or upstream of callose synthesis. To establish the hierarchy of signaling at plasmodesmata and characterize points of convergence in microbe elicitor-triggered signaling, we profiled the dependence of plasmodesmal responses triggered by different elicitors on a range of plasmodesmal signaling machinery. We identified that, like chitin, flg22 signals via RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) to induce plasmodesmal closure. Further, we found that PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1), PDLP5, and CALLOSE SYNTHASE 1 (CALS1) are common to microbe- and salicylic acid (SA)-triggered responses, identifying PDLPs as a candidate signaling nexus. To understand how PDLPs relay a signal to CALS1, we screened for PDLP5 interactors and found NON-RACE SPECIFIC DISEASE RESISTANCE/HIN1 HAIRPIN-INDUCED-LIKE protein 3 (NHL3), which is also required for chitin-, flg22- and SA-triggered plasmodesmal responses and PDLP-mediated activation of callose synthesis. We conclude that a PDLP-NHL3 complex acts as an integrating node of plasmodesmal signaling cascades, transmitting multiple immune signals to activate CALS1 and plasmodesmata closure.

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.)

Plasmodesmata are cytosolic bridges, lined by the plasma membrane and traversed by endoplasmic reticulum; plasmodesmata connect cells and tissues, and are critical for many aspects of plant biology. While plasmodesmata are notoriously difficult to extract, tissue fractionation and proteomic analyses can yield valuable knowledge of their composition. Here we have generated two novel proteomes to expand tissue and taxonomic representation of plasmodesmata: one from mature Arabidopsis leaves and one from the moss Physcomitrium patens, and leveraged these and existing data to perform a comparative analysis to identify evolutionarily conserved protein families that are associated with plasmodesmata. Thus, we identified β-1,3-glucanases, C2 lipid-binding proteins, and tetraspanins as core plasmodesmal components that probably serve as essential structural or functional components. Our approach has not only identified elements of a conserved plasmodesmal proteome, but also demonstrated the added power offered by comparative analysis for recalcitrant samples. Conserved plasmodesmal proteins establish a basis upon which ancient plasmodesmal function can be further investigated to determine the essential roles these structures play in multicellular organism physiology in the green lineages., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Arabinogalactan proteins (AGPs) are a plant-specific family of extracellular proteoglycans characterized by large and complex galactose-rich polysaccharide chains. Functional elucidation of AGPs, however, has been hindered by the high degree of redundancy of AGP genes. To uncover as yet unexplored roles of AGPs in Arabidopsis, a mutant of Hyp O-galactosyltransferase (HPGT), a critical enzyme that catalyzes the common initial step of Hyp-linked arabinogalactan chain biosynthesis, was used. Here we show, using the hpgt1,2,3 triple mutant, that a reduction in functional AGPs leads to a stomatal patterning defect in which two or more stomata are clustered together. This defect is attributed to increased and dysregulated symplastic transport following changes in plasmodesmata structure, such that highly permeable complex branched plasmodesmata with cavities in branching parts increased in the mutant. We also found that the hpgt1,2,3 mutation causes a reduction of cellulose in the cell wall and accumulation of pectin, which controls cell wall porosity. Our results highlight the importance of AGPs in the correct biogenesis of plasmodesmata, possibly acting through the regulation of cell wall properties surrounding the plasmodesmata., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)