Intercellular protein movement: deciphering the language of development.

Development in multicellular organisms requires the coordinated production of a large number of specialized cell types through sophisticated signaling mechanisms. Non-cell-autonomous signals are one of the key mechanisms by which organisms coordinate development. In plants, intercellular movement of transcription factors and other mobile signals, such as hormones and peptides, is essential for normal development. Through a combination of different approaches, a large number of non-cell-autonomous signals that control plant development have been identified. We review some of the transcriptional regulators that traffic between cells, as well as how changes in symplasmic continuity affect and are affected by development. We also review current models for how mobile signals move via plasmodesmata and how movement is inhibited. Finally, we consider challenges in and new tools for studying protein movement.

PLASMODESMATA-LOCATED PROTEIN 6 regulates plasmodesmal function in Arabidopsis vasculature.

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.

Targeting of plasmodesmal proteins requires unconventional signals.

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.

A PDLP-NHL3 complex integrates plasmodesmal immune signaling cascades.

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.

P3N-PIPO but not P3 is the avirulence determinant in melon carrying the Wmr resistance against watermelon mosaic virus, although they contain a common genetic determinant.

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.

The CELL NUMBER REGULATOR FW2.2 protein regulates cell-to-cell communication in tomato by modulating callose deposition at plasmodesmata.

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.

Symplasmic phloem loading and subcellular transport in storage roots are key factors for carbon allocation in cassava.

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.

Plasmodesmata mediate cell-to-cell transport of brassinosteroid hormones.

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.

Plasmodesmata play a key role in leaf vein patterning.

Leaf veins provide a vital transport route in plants. The formation of leaf vein patterns has fascinated many scientists. In PLOS Biology, Linh and Scarpella reveal that transport through plasmodesmata plays a key role in vein patterning.

Leaf vein patterning is regulated by the aperture of plasmodesmata intercellular channels.

To form tissue networks, animal cells migrate and interact through proteins protruding from their plasma membranes. Plant cells can do neither, yet plants form vein networks. How plants do so is unclear, but veins are thought to form by the coordinated action of the polar transport and signal transduction of the plant hormone auxin. However, plants inhibited in both pathways still form veins. Patterning of vascular cells into veins is instead prevented in mutants lacking the function of the GNOM (GN) regulator of auxin transport and signaling, suggesting the existence of at least one more GN-dependent vein-patterning pathway. Here we show that in Arabidopsis such a pathway depends on the movement of auxin or an auxin-dependent signal through plasmodesmata (PDs) intercellular channels. PD permeability is high where veins are forming, lowers between veins and nonvascular tissues, but remains high between vein cells. Impaired ability to regulate PD aperture leads to defects in auxin transport and signaling, ultimately leading to vein patterning defects that are enhanced by inhibition of auxin transport or signaling. GN controls PD aperture regulation, and simultaneous inhibition of auxin signaling, auxin transport, and regulated PD aperture phenocopies null gn mutants. Therefore, veins are patterned by the coordinated action of three GN-dependent pathways: auxin signaling, polar auxin transport, and movement of auxin or an auxin-dependent signal through PDs. Such a mechanism of tissue network formation is unprecedented in multicellular organisms.

Arabinogalactan protein polysaccharide chains are required for normal biogenesis of plasmodesmata.

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.

Arabidopsis callus grafts: a system to study symplasmic intercellular macromolecular transport and gene silencing spread.

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.

Assaying Effector Cell-to-Cell Mobility in Plant Tissues Identifies Hypermobility and Indirect Manipulation of Plasmodesmata.

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.

Facilitation of Symplastic Effector Protein Mobility by Paired Effectors Is Conserved in Different Classes of Fungal Pathogens.

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 FolSix5 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.

Comparing Methods for Detection and Quantification of Plasmodesmal Callose in Nicotiana benthamiana Leaves During Defense Responses.

Callose, a ß-(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.

Plasmodesmata dynamics in bryophyte model organisms: secondary formation and developmental modifications of structure and function.

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.

Plasmodesmata-associated Flotillin positively regulates broad-spectrum virus cell-to-cell trafficking.

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.

Plasmodesmata and intercellular molecular traffic control.

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.

Plasmodesmal connectivity in C4 Gynandropsis gynandra is induced by light and dependent on photosynthesis.

In leaves of C4 plants, the reactions of photosynthesis become restricted between two compartments. Typically, this allows accumulation of C4 acids in mesophyll (M) cells and subsequent decarboxylation in the bundle sheath (BS). In C4 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 C4 dicotyledons also show this enhanced plasmodesmal connectivity and so whether this is a general requirement for C4 photosynthesis is not clear. How M and BS cells in C4 leaves become highly connected is also not known. We investigated these questions using 3D- and 2D-electron microscopy on the C4 dicotyledon Gynandropsis gynandra as well as phylogenetically close C3 relatives. The M-BS interface of C4 G. gynandra showed higher plasmodesmal frequency compared with closely related C3 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 C4 G. gynandra.

Geminivirus C5 proteins mediate formation of virus complexes at plasmodesmata for viral intercellular movement.

Movement proteins (MPs) encoded by plant viruses deliver viral genomes to plasmodesmata (PD) to ensure intracellular and intercellular transport. However, how the MPs encoded by monopartite geminiviruses are targeted to PD is obscure. Here, we demonstrate that the C5 protein of tomato yellow leaf curl virus (TYLCV) anchors to PD during the viral infection following trafficking from the nucleus along microfilaments in Nicotiana benthamiana. C5 could move between cells and partially complement the traffic of a movement-deficient turnip mosaic virus (TuMV) mutant (TuMV-GFP-P3N-PIPO-m1) into adjacent cells. The TYLCV-C5 null mutant (TYLCV-mC5) attenuates viral pathogenicity and decreases viral DNA and protein accumulation, and ectopic overexpression of C5 enhances viral DNA accumulation. Interaction assays between TYLCV-C5 and the other eight viral proteins described in TYLCV reveal that C5 associates with C2 in the nucleus and with V2 in the cytoplasm and at PD. The V2 protein is mainly localized in the nucleus and cytoplasmic granules when expressed alone; in contrast, V2 forms small punctate granules at PD when co-expressed with C5 or in TYLCV-infected cells. The interaction of V2 and C5 also facilitates their nuclear export. Furthermore, C5-mediated PD localization of V2 is conserved in two other geminiviruses. Therefore, this study solves a long-sought-after functional connection between PD and the geminivirus movement and improves our understanding of geminivirus-encoded MPs and their potential cellular and molecular mechanisms.