Size Exclusion Limit Of Plasmodesmata Research Articles

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.

The primary function of Six5 of Fusarium oxysporum is to facilitate Avr2 activity by together manipulating the size exclusion limit of plasmodesmata.

Pathogens produce effector proteins to manipulate their hosts. While most effectors act autonomously, some fungal effectors act in pairs and rely on each other for function. During the colonization of the plant vasculature, the root-infecting fungus Fusarium oxysporum (Fo) produces 14 so-called Secreted in Xylem (SIX) effectors. Two of these effector genes, Avr2 (Six3) and Six5, form a gene pair on the pathogenicity chromosome of the tomato-infecting Fo strain. Avr2 has been shown to suppress plant defense responses and is required for full pathogenicity. Although Six5 and Avr2 together manipulate the size exclusion limit of plasmodesmata to facilitate cell-to-cell movement of Avr2, it is unclear whether Six5 has additional functions as well. To investigate the role of Six5, we generated transgenic Arabidopsis lines expressing Six5. Notably, increased susceptibility during the early stages of infection was observed in these Six5 lines, but only to Fo strains expressing Avr2 and not to wild-type Arabidopsis-infecting Fo strains lacking this effector gene. Furthermore, neither PAMP-triggered defense responses, such as ROS accumulation and callose deposition upon treatment with Flg22, necrosis and ethylene-inducing peptide 1-like protein (NLP), or chitosan, nor susceptibility to other plant pathogens, such as the bacterium Pseudomonas syringae or the fungus Verticilium dahlia, were affected by Six5 expression. Further investigation of the ability of the Avr2/Six5 effector pair to manipulate plasmodesmata (PD) revealed that it not only permits cell-to-cell movement of Avr2, but also facilitates the movement of two additional effectors, Six6 and Six8. Moreover, although Avr2/Six5 expands the size exclusion limit of plasmodesmata (i.e., gating) to permit the movement of a 2xFP fusion protein (53 kDa), a larger variant, 3xFP protein (80 kDa), did not move to the neighboring cells. The PD manipulation mechanism employed by Avr2/Six5 did not involve alteration of callose homeostasis in these structures. In conclusion, the primary function of Six5 appears to function together with Avr2 to increase the size exclusion limit of plasmodesmata by an unknown mechanism to facilitate cell-to-cell movement of Fo effectors.

Virus Genome-Based Reporter for Analyzing Viral Movement Proteins and Plasmodesmata Permeability.

Plant virus movement proteins (MPs) mediate cell-to-cell movement of the virus genome through plasmodesmata (PD). MPs target PD to increase their size exclusion limit (SEL), and this MP function is essential for virus intercellular trafficking. In this chapter, we describe the use of a Potato virus X genome-derived reporter for agroinfiltration-based identification of virus genome-encoded MPs and analysis of the ability of individual viral MPs or plant proteins to increase the PD SEL.

Analysis of a novel mutant allele of GSL8 reveals its key roles in cytokinesis and symplastic trafficking in Arabidopsis

BackgroundPlant cell walls are mainly composed of polysaccharides such as cellulose and callose. Callose exists at a very low level in the cell wall; however, it plays critical roles at different stages of plant development as well as in defence against unfavorable conditions. Callose is accumulated at the cell plate, at plasmodesmata and in male and female gametophytes. Despite the important roles of callose in plants, the mechanisms of its synthesis and regulatory properties are not well understood.ResultsCALLOSE SYNTHASE (CALS) genes, also known as GLUCAN SYNTHASE-LIKE (GSL), comprise a family of 12 members in Arabidopsis thaliana. Here, we describe a new allele of GSL8 (named essp8) that exhibits pleiotropic seedling defects. Reduction of callose deposition at the cell plates and plasmodesmata in essp8 leads to ectopic endomitosis and an increase in the size exclusion limit of plasmodesmata during early seedling development. Movement of two non-cell-autonomous factors, SHORT ROOT and microRNA165/6, both required for root radial patterning during embryonic root development, are dysregulated in the primary root of essp8. This observation provides evidence for a molecular mechanism explaining the gsl8 root phenotype. We demonstrated that GSL8 interacts with PLASMODESMATA-LOCALIZED PROTEIN 5, a β-1,3-glucanase, and GSL10. We propose that they all might be part of a putative callose synthase complex, allowing a concerted regulation of callose deposition at plasmodesmata.ConclusionAnalysis of a novel mutant allele of GSL8 reveals that GSL8 is a key player in early seedling development in Arabidopsis. GSL8 is required for maintaining the basic ploidy level and regulating the symplastic trafficking. Callose deposition at plasmodesmata is highly regulated and occurs through interaction of different components, likely to be incorporated into a callose biosynthesis complex. We are providing new evidence supporting an earlier hypothesis that GSL8 might have regulatory roles apart from its enzymatic function in plasmodesmata regulation.

Lipid Raft, Regulator of Plasmodesmal Callose Homeostasis.

The specialized plasma membrane microdomains known as lipid rafts are enriched by sterols and sphingolipids. Lipid rafts facilitate cellular signal transduction by controlling the assembly of signaling molecules and membrane protein trafficking. Another specialized compartment of plant cells, the plasmodesmata (PD), which regulates the symplasmic intercellular movement of certain molecules between adjacent cells, also contains a phospholipid bilayer membrane. The dynamic permeability of plasmodesmata (PDs) is highly controlled by plasmodesmata callose (PDC), which is synthesized by CALLOSE SYNTHASES (CalS) and degraded by β-1,3-GLUCANASES (BGs). In recent studies, remarkable observations regarding the correlation between lipid raft formation and symplasmic intracellular trafficking have been reported, and the PDC has been suggested to be the regulator of the size exclusion limit of PDs. It has been suggested that the alteration of lipid raft substances impairs PDC homeostasis, subsequently affecting PD functions. In this review, we discuss the substantial role of membrane lipid rafts in PDC homeostasis and provide avenues for understanding the fundamental behavior of the lipid raft–processed PDC.

Plasmodesmata: channels for viruses on the move.

The symplastic communication network established by plasmodesmata (PD) and connected phloem provides an essential pathway for spatiotemporal intercellular signaling in plant development but is also exploited by viruses for moving their genomes between cells in order to infect plants systemically. Virus movement depends on virus-encoded movement proteins (MPs) that target PD and therefore represent important keys to the cellular mechanisms underlying the intercellular trafficking of viruses and other macromolecules. Viruses and their MPs have evolved different mechanisms for intracellular transport and interaction with PD. Some viruses move from cell to cell by interacting with cellular mechanisms that control the size exclusion limit of PD whereas other viruses alter the PD architecture through assembly of specialized transport structures within the channel. Some viruses move between cells in the form of assembled virus particles whereas other viruses may interact with nucleic acid transport mechanisms to move their genomes in a non-encapsidated form. Moreover, whereas several viruses rely on the secretory pathway to target PD, other viruses interact with the cortical endoplasmic reticulum and associated cytoskeleton to spread infection. This chapter provides an introduction into viruses and their role in studying the diverse cellular mechanisms involved in intercellular PD-mediated macromolecular trafficking.

Analysis of the conductivity of plasmodesmata by microinjection.

Pressure microinjection can be used to introduce fluorescent dyes and labeled macromolecules into single cells. The method allows measuring transport activity of macromolecules such as proteins and RNA molecules within and between cells. Routinely, plant mesophyll cells are injected with fluorescent dextran molecules of specific sizes to measure an increase of the size exclusion limit of plasmodesmata in the presence of a co-injected or expressed protein. The mobility of a macromolecule can also be addressed directly by injecting a recombinant protein that itself is labeled with fluorescent dye and following its transport to neighboring cells. This chapter describes a pressure microinjection protocol successfully applied to Nicotiana leaves. This protocol requires basic skills and experience in handling a microscope equipped with an imaging system, a micromanipulator, and a microinjection system attached to an upright microscope. Using this equipment, a trained person can inject approximately 10-20 mesophyll cells per hour.

Plasmodesmata: channels for intercellular signaling during plant growth and development.

Plants have evolved strategies for short- and long-distance communication to coordinate plant development and to adapt to changing environmental conditions. Plasmodesmata (PD) are intercellular nanochannels that provide an effective pathway for both selective and nonselective movement of various molecules that function in diverse biological processes. Numerous non-cell-autonomous proteins (NCAP) and small RNAs have been identified that have crucial roles in cell fate determination and organ patterning during development. Both the density and aperture size of PD are developmentally regulated, allowing formation of spatial symplastic domains for establishment of tissue-specific developmental programs. The PD size exclusion limit (SEL) is controlled by reversible deposition of callose, as well as by some PD-associated proteins. Although a large number of PD-associated proteins have been identified, many of their functions remain unknown. Despite the fact that PD are primarily membranous structures, surprisingly very little is known about their lipid composition. Thus, future studies in PD biology will provide deeper insights into the high-resolution structure and tightly regulated functions of PD and the evolution of PD-mediated cell-to-cell communication in plants.

Plasmodesmata formation and cell-to-cell transport are reduced in decreased size exclusion limit 1 during embryogenesis in Arabidopsis.

In plants, plasmodesmata (PD) serve as channels for micromolecular and macromolecular cell-to-cell transport. Based on structure, PD in immature tissues are classified into two types, simple and branched (X- and Y-shaped) or twinned. The maximum size of molecules capable of PD transport defines PD aperture, known as the PD size exclusion limit. Here we report an Arabidopsis mutation, decreased size exclusion limit1 (dse1), that exhibits reduced cell-to-cell transport of the small (524 Da) fluorescent tracer 8-hydroxypyrene-1,3,6-trisulfonic acid at the midtorpedo stage of embryogenesis. Correspondingly, the fraction of X- and Y-shaped and twinned PD was reduced in dse1 embryos compared with WT embryos at this stage, suggesting that the frequency of PD is related to transport capability. dse1 is caused by a point mutation in At4g29860 (previously termed TANMEI) at the last donor splice site of its transcript, resulting in alternative splicing in both the first intron and the last intron. AtDSE1 is a conserved eukaryotic 386-aa WD-repeat protein critical for Arabidopsis morphogenesis and reproduction. Similar to its homologs in mouse, null mutants are embryo-lethal. The weak loss-of-function mutant dse1 exhibits pleiotropic phenotypes, including retarded vegetative growth, delayed flowering time, dysfunctional male and female organs, and delayed senescence. Finally, silencing of DSE1 in Nicotiana benthamiana leaves leads to reduced movement of GFP fused to tobacco mosaic virus movement protein. Thus, DSE1 is important for regulating PD transport between plant cells.

Viral cell-to-cell movement requires formation of cortical punctate structures containing Red clover necrotic mosaic virus movement protein

Movement protein (MP) of Red clover necrotic mosaic virus (RCNMV) forms punctate structures on the cortical endoplasmic reticulum (ER) of Nicotiana benthamiana cells, which are associated with viral RNA1 replication (Kaido et al., Virology 395, 232–242. 2009). We investigated the significance of ER-targeting by MP during virus movement from cell to cell, by analyzing the function of a series of MPs with varying length deletions at their C-terminus, either fused or not fused with green fluorescent protein (GFP). The C-terminal 70 amino acids were crucial to ER-localization of MP-GFP and cell-to-cell movement of the recombinant virus encoding it. However, C-terminal deletion did not affect MP functions, such as increasing the size exclusion limit of plasmodesmata, single-stranded RNA binding in vitro, and MP interacting in vivo. We discuss the possible role of this MP region in virus movement from cell to cell.

Cucumber Mosaic VirusMovement Protein Severs Actin Filaments to Increase the Plasmodesmal Size Exclusion Limit in Tobacco

Plant viral movement proteins (MPs) enable viruses to pass through cell walls by increasing the size exclusion limit (SEL) of plasmodesmata (PD). Here, we report that the ability of Cucumber mosaic virus (CMV) MP to increase the SEL of the PD could be inhibited by treatment with the actin filament (F-actin)-stabilizing agent phalloidin but not by treatment with the F-actin-destabilizing agent latrunculin A. In vitro studies showed that CMV MP bound globular and F-actin, inhibited actin polymerization, severed F-actin, and participated in plus end capping of F-actin. Analyses of two CMV MP mutants, one with and one without F-actin severing activities, demonstrated that the F-actin severing ability was required to increase the PD SEL. Furthermore, the Tobacco mosaic virus MP also exhibited F-actin severing activity, and its ability to increase the PD SEL was inhibited by treatment with phalloidin. Our data provide evidence to support the hypothesis that F-actin severing is required for MP-induced increase in the SEL of PD. This may have broad implications in the study of the mechanisms of actin dynamics that regulate cell-to-cell transport of viral and endogenous proteins.

Impact of RNA Virus Infection on Plant Cell Function and Evolution

Viruses are obligate symbionts that tightly interact with their hosts to complete their life cycle. Each infected cell is confronted with the accumulation of viral products and activities that have evolved to support the replication and spread of the virus in the context of host cell functions and defense responses. Tobacco mosaic virus encodes replicase proteins and coat protein, to replicate and protect the RNA genome, and a movement protein (MP) that binds viral RNA and manipulates the size exclusion limit of plasmodesmata to facilitate the spread of the viral genomic RNA (vRNA). The MP and replicase also interfere with the cellular RNA silencing machinery that influences plant gene expression and development. Moreover, virus-infected cells stimulate the production of a systemic signal ahead of the virus front that triggers genomic recombination leading to heritable genetic changes. Thus viruses can interact with their hosts through diverse molecular interactions. Given the high mutation rate of viruses, these interactions have implications for evolutionary processes and adaptations at the virus-host interface that may contribute to eukaryotic evolution.

Cell-to-cell movement of the CAPRICE protein inArabidopsisroot epidermal cell differentiation

CAPRICE (CPC), a small, R3-type Myb-like protein, is a positive regulator of root hair development in Arabidopsis. Cell-to-cell movement of CPC is important for the differentiation of epidermal cells into trichoblasts (root hair cells). CPC is transported from atrichoblasts (hairless cells), where it is expressed, to trichoblasts, and generally accumulates in their nuclei. Using truncated versions of CPC fused to GFP, we identified a signal domain that is necessary and sufficient for CPC cell-to-cell movement. This domain includes the N-terminal region and a part of the Myb domain. Amino acid substitution experiments indicated that W76 and M78 in the Myb domain are critical for targeted transport, and that W76 is crucial for the nuclear accumulation of CPC:GFP. To evaluate the tissue-specificity of CPC movement, CPC:GFP was expressed in the stele using the SHR promoter and in trichoblasts using the EGL3 promoter. CPC:GFP was able to move from trichoblasts to atrichoblasts but could not exit from the stele, suggesting the involvement of tissue-specific regulatory factors in the intercellular movement of CPC. Analyses with a secretion inhibitor, Brefeldin A, and with an rhd3 mutant defective in the secretion process in root epidermis suggested that intercellular CPC movement is mediated through plasmodesmata. Furthermore, the fusion of CPC to tandem-GFPs defined the capability of CPC to increase the size exclusion limit of plasmodesmata.

Two Plant–Viral Movement Proteins Traffic in the Endocytic Recycling Pathway

Many plant viruses exploit a conserved group of proteins known as the triple gene block (TGB) for cell-to-cell movement. Here, we investigated the interaction of two TGB proteins (TGB2 and TGB3) of Potato mop-top virus (PMTV), with components of the secretory and endocytic pathways when expressed as N-terminal fusions to green fluorescent protein or monomeric red fluorescent protein (mRFP). Our studies revealed that fluorophore-labeled TGB2 and TGB3 showed an early association with the endoplasmic reticulum (ER) and colocalized in motile granules that used the ER-actin network for intracellular movement. Both proteins increased the size exclusion limit of plasmodesmata, and TGB3 accumulated at plasmodesmata in the absence of TGB2. TGB3 contains a putative Tyr-based sorting motif, mutations in which abolished ER localization and plasmodesmatal targeting. Later in the expression cycle, both fusion proteins were incorporated into vesicular structures. TGB2 associated with these structures on its own, but TGB3 could not be incorporated into the vesicles in the absence of TGB2. Moreover, in addition to localization to the ER and motile granules, mRFP-TGB3 was incorporated into vesicles when expressed in PMTV-infected epidermal cells, indicating recruitment by virus-expressed TGB2. The TGB fusion protein-containing vesicles were labeled with FM4-64, a marker for plasma membrane internalization and components of the endocytic pathway. TGB2 also colocalized in vesicles with Ara7, a Rab5 ortholog that marks the early endosome. Protein interaction analysis revealed that recombinant TGB2 interacted with a tobacco protein belonging to the highly conserved RME-8 family of J-domain chaperones, shown to be essential for endocytic trafficking in Caenorhabditis elegans and Drosophila melanogaster. Collectively, the data indicate the involvement of the endocytic pathway in viral intracellular movement, the implications of which are discussed.

Plasmodesmata and the control of symplastic transport

In 1879 Eduard Tangle discovered cytoplasmic connections between cells in the cotyledons of Strychnos nuxvomica , which he interpreted to be protoplasmic contacts. This led him to hypothesize that ‘the protoplasmic bodies . . . are united by thin strands passing through connecting ducts in the walls, which put the cells into connection with each other and so unite them to an entity of higher order’ (Carr 1976). This challenged the then current view that cells functioned as autonomous units. It was after much research in many other species and cell types that Strasburger, in 1901, named these structures plasmodesmata (Carr 1976). During the division and differentiation of meristematic cells, plasmodesmata are formed across each developing cell plate, allowing cytoplasmic and endomembrane continuity to occur between all daughter cells, and ultimately, between all cells in a developing tissue (Mezitt & Lucas 1996). Those plasmodesmata that form during cell division are termed primary plasmodesmata (Jones 1976). Those that form de novo across existing cell walls are called secondary plasmodesmata (Ehlers & Kollmann 2001). The formation of secondary plasmodesmata allows cells to increase their potential for molecular trafficking and allows connections to be created between cells that are not related cytokinetically. As cells expand and differentiate, their fate determines the extent to which their cytoplasmic connectivity to other cells is maintained (Mezitt & Lucas 1996). Some cell types, such as those of the leaf mesophyll, remain closely connected to their neighbours, and may even lay down additional plasmodesmata to increase the continuity (Ding et al . 1992a). In other areas of the plant, for instance in vascular tissue, certain cells greatly reduce the number of plasmodesmata in their adjoining walls (Gamalei 1989). In this way, the cytoplasmic continuity can be altered depending on the tissue type (Botha & Evert 1988; Brown et al . 1995). However, although reductions in the number of plasmodesmata are common, only guard cells surrounding stomata (Erwee, Goodwin & van Bel 1985; Palevitz & Hepler 1985) and differentiating xylem elements (Lachaud & Maurousset 1996) lose all symplastic connections at maturity. In all other cells, some degree of intercellular connection is maintained. This plasmodesmal continuum that potentially exists throughout the whole plant is termed the symplast (Munch 1930). However, the symplast is not the open continuum that Munch originally hypothesized, but is divided into functional domains, each tightly regulated by different forms of plasmodesmata (Erwee & Goodwin 1985; Ehlers & Kollmann 2001). Plasmodesmata are now thought of as fluid, dynamic structures that can be modified both structurally and functionally to cope with the requirements of specific cells and tissues.

Immigration control in plant cells

Plasmodesmata are thought to be key in plant cell–cell communication. These narrow channels cross plant cell walls and connect the cytoplasm of a cell with that of its neighbours. The structure of plasmodesmata has been well characterized, but details of their function and regulation are lacking. A significant feature of plasmodesmata is their size exclusion limit (SEL), which is the maximum size of molecule that can diffuse freely through from one cell to the next. SELs vary spatially and temporally, and they are used to define ‘symplastic domains’; that is, tissues or groups of cells that appear to have functionally equivalent plasmodesmata, allowing certain molecules to move freely throughout that group of cells. Symplastic domains are thought to have important roles in plant development and plant defense.To examine how these domains are set up and regulated, Kim et al. [1xIdentification of a developmental transition in plasmodesmatal function during embryogenesis in Arabidopsis thaliana. Kim, I et al. Development. 2002; 129: 1261–1272PubMedSee all References][1] incubated Arabidopsis thaliana embryos in media containing fluorescent tracer molecules such as HPTS (524 Da) or 10-kDa F-dextran. They identified a new symplastic transition phase, around mid-torpedo stage. Both HPTS and 10- kDa F-dextran could move throughout the whole embryo till the early torpedo stage, but then the SEL of cells seemed to decrease so that 10-kDa F-dextran was no longer transported, but HPTS was still able to move freely throughout.They then screened for mutants in which plasmodesmatal transport was aberrant. They focused their search on embryo defective mutants, reasoning that any significant change to plasmodesmata function would be likely to affect the plant early in development. The screen identified two genes that appear to be involved in regulating the decrease in SEL at the mid-torpedo stage, ISE1 and ISE2 (for ‘increased size exclusion limit of plasmodesmata’). When these genes were mutated, the restricted movement of 10-kDa F-dextrans appeared to be abolished. The mutations caused no other morphological defects (including no obvious effects on plasmodesmata structure), except for a slight developmental retardation and abnormal root hair production. These effects could be a result of failure to restrict the movement of certain molecules. Another possibility is that the morphological changes are the cause of the plasmodesmatal defects, but this is less likely as the authors identified nearly 5000 other mutant lines that had morphological defects but no plasmodesmatal defects.The evidence suggests that the ISE genes are required purely for the regulation of plasmodesmata SELs. If this is so, then their characterization could provide insight into plasmodesmatal regulation and the establishment of symplastic domains. A key question is the level at which the ISE genes function. Do their encoded products interact directly with plasmodesmata, or do they work at a higher level, regulating the change in SEL throughout the whole embryo, for example?

Identification of a developmental transition in plasmodesmatal function during embryogenesis in Arabidopsis thaliana.

Plasmodesmata provide routes for communication and nutrient transfer between plant cells by interconnecting the cytoplasm of adjacent cells. A simple fluorescent tracer loading assay was developed to monitor patterns of cell-to-cell transport via plasmodesmata specifically during embryogenesis. A developmental transition in plasmodesmatal size exclusion limit was found to occur at the torpedo stage of embryogenesis in Arabidopsis; at this time, plasmodesmata are down-regulated, allowing transport of small (approx. 0.5 kDa) but not large (approx. 10 kDa) tracers. This assay system was used to screen for embryo-defective mutants, designated increased size exclusion limit of plasmodesmata (ise), that maintain dilated plasmodesmata at the torpedo stage. The morphology of ise1 and ise2 mutants discussed here resembled that of the wild-type during embryo development, although the rate of their embryogenesis was slower. The ISE1 gene was mapped to position 13 cM on chromosome I using PCR-based biallelic markers. ise2 was found to be allelic to the previously characterized mutant emb25 which maps to position 100 cM on chromosome I. The results presented have implications for intercellular signaling pathways that regulate embryonic development, and furthermore represent the first attempt to screen directly for mutants of Arabidopsis with altered size exclusion limit of plasmodesmata.

Local expression of enzymatically active class I beta-1, 3-glucanase enhances symptoms of TMV infection in tobacco.

Mutant tobacco plants deficient for class I beta-1,3-glucanase (GLU I) are decreased in their susceptibility to virus infection. This is correlated with delayed virus spread, a reduction in the size exclusion limit of plasmodesmata and increased cell-wall deposition of the beta-1,3-glucan callose. To further investigate a role of GLU I during cell-to-cell movement of virus infection, we inserted the GLU I coding sequence into TMV for overexpression in infected cells. Compared with the size of local lesions produced on plants infected with virus expressing either an enzymatically inactive GLU I or a frameshift mutant of the gene, the size of local lesions caused by infection with virus expressing active GLU I was consistently increased. Viruses expressing antisense GLU I constructs led to lesions of decreased size. Similar effects were obtained for virus spread using plants grown at 32 degrees C to block the hypersensitive response. Together, these results indicate that enzymatically active GLU I expressed in cells containing replicating virus can increase cell-to-cell movement of virus. This supports the view that GLU I induced locally during infection helps to promote cell-to-cell movement of virus by hydrolyzing callose. Moreover, our results provide the first direct evidence that a biological function of a plant beta-1,3-glucanase depends on its catalytic activity.

Structure and function of plasmodesmata

Little is known about the molecular architecture of plasmodesmata. Electron micrographs of plasmodesmata, although showing remarkably consistent features, do not reveal the dynamic nature of plasmodesmata. In addition, the generally accepted size exclusion limit of plasmodesmata of 1 kDa as determined by the microinjection of fluorescent probes, is not an accurate measure of the size of molecules that can move from cell to cell. In some cases, plasmodesmata allow the non-selective movement of proteins up to 50 kDa, whereas others allow the selective transport of proteins such as transcription factors. Insights to the molecular architecture of plasmodesmata and the mechanism of cell-to-cell transport can be gained by the examination of the type of molecules and conditions under which selective and passive movement of molecules occurs.

Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues.

Macromolecular trafficking within the sieve element-companion cell complex, phloem unloading, and post-phloem transport were studied using the jellyfish green fluorescent protein (GFP). The GFP gene was expressed in Arabidopsis and tobacco under the control of the AtSUC2 promoter. In wild-type Arabidopsis plants, this promoter regulates expression of the companion cell-specific AtSUC2 sucrose-H+ symporter gene. Analyses of the AtSUC2 promoter-GFP plants demonstrated that the 27-kD GFP protein can traffic through plasmodesmata from companion cells into sieve elements and migrate within the phloem. With the stream of assimilates, the GFP is partitioned between different sinks, such as petals, root tips, anthers, funiculi, or young rosette leaves. Eventually, the GFP can be unloaded symplastically from the phloem into sink tissues, such as the seed coat, the anther connective tissue, cells of the root tip, and sink leaf mesophyll cells. In all of these tissues, the GFP can traffic cell to cell by symplastic post-phloem transport. The presented data show that plasmodesmata of the sieve element-companion cell complex, as well as plasmodesmata into and within the analyzed sinks, allow trafficking of the 27-kD nonphloem GFP protein. The data also show that the size exclusion limit of plasmodesmata can change during organ development. The results are also discussed in terms of the phloem mobility of assimilates and of small, low molecular weight companion cell proteins.