Plasmodesmata transport of GFP and GFP fusions requires little energy and transitions during leaf expansion

Plasmodesmata (Pd) are symplastic channels between neighboring plant cells and are key in plant cell-cell signaling. Viruses of proteins, nucleic acids, and a wide range of signaling macromolecules move across Pd. Protein transport Pd is regulated by development and biotic signals. Recent investigations utilizing the Arrhenius equation or Coefficient of conductivity showed that fundamental energetic measurements used to describe transport of proteins across membrane pores or the nuclear pore can also apply to protein movement across Pd. As leaves continue to expand, Pd transport of proteins declines which may result from changes in cell volume, Pd density or Pd structure.Key words: plasmodesmata, diffusion, GFP, viral transport, PVX, triple gene blockResearchers have argued for the last decade that movement of proteins and other macromolecules across Pd is regulated by development, stress and biotic signals. There are four current models describing different mechanisms of Pd transport. First is the non cell autonomous protein (NCAP) pathway that carries ribonucleoprotein complexes across Pd. NCAPs often carry RNA in a ribonucleoprotein complex to the Pd.^1–4 This mode of transport involved targeted movement, meaning that a set of proteins must dock within the Pd to gate it open to enable transfer between cells. Proteins which are normally too large to move across Pd can gate open Pd to enable its own transfer into neighboring cells. This is contrasted by nontargetted movement, which is passive movement of proteins that are sufficiently small enough to pass between cells.^5,6 The green fluorescent protein (GFP) has been described as a protein whose movement is non-targeted, meaning that it can diffuse across Pd. Reasons that we do not see continuous movement of small proteins between cells include protein compartmentalization or subcellular targeting signals. For example proteins may be synthesized and modified via the ER and Golgi networks and then transferred into vesicles and transported within cells to their destination. These proteins would not be free in the cytosol for diffusion across Pd. Alternatively, proteins which have dominant subcellular targeting signals which direct them to certain organelles such as the nucleus, peroxisome, or other destination would not be free to move across Pd.^5,6 A third model represents proteins in the ER that move laterally along the membrane or through the ER lumen into neighboring cells. This transport is quite rapid and investigations are ongoing to determine how this is regulated.^7–11 Finally, there is vesicle transport which deliver cargo to Pd.^12,13 The origin of these vesicles is still under investigation. Much more research has been accomplished toward defining non-targeted movement and the NCAP pathway while the ER and vesicle transport pathways are only recently described and very little is known about the regulatory mechanisms underlying these pathways.Pd permeability is governed in part by architecture, but also by key regulatory factors that determine Pd conductivity. Factors such as mysoin VIII, actin and calreticulin were identified in Pd which likely regulate expansion and contraction.^14–19 In addition calcium, ATP and plant hormones can downregulate Pd permeability during development and stress.^20,21 The tools for measuring Pd permeability has been to study the transport of fluorescently tagged proteins, fluorescent dextran beads, GFP or GFP fusions following microinjection or biolistic delivery to the cytoplasm of one cell.^22–26 Then video imaging or captured still images at select time intervals are used to characterize Pd transport. Until recently researchers quantified movement by the frequency they observed a certain type of movement. Therefore our ability to describe Pd permeability has been limited.Evidence that ATP impacts Pd conductivity has led investigations to explore the energy requirements for macromolecular transport across Pd. By understanding the energy requirements for transport of various proteins and nucleic acids we can better characterize passive or active transport processes. Toward this end two recent studies detailed quantitative approaches that can be employed to describe the developmental and energy requirements cell-to-cell transport of cytosolic proteins. Both papers used biolistic bombardment to deliver plasmids expressing GFP or GFP fusions to tobacco leaf epidermal cells and then captured still images of GFP fluorescence in neighboring cells. We employed the Arrhenius equation to characterize transport of GFP or GFP fused to the Potato virus X (PVX) TGBp1 movement protein. PVX TGBp1 was selected to compare with GFP alone since it is known to gate open Pd and has ATPase activity.⁴⁵ We predicted that the abilities of GFP alone and GFP-TGBp1 to move across Pd might be different and were surprised to learn that the energy for transport of both proteins was similar. This project established the principle that GFP and GFP-TGBp1 transport is temperature dependent showing a linear relationship between protein movement and the temperatures at which leaves were incubated.Green fluorescent sites on bombarded leaves were scored for the movement or no movement. Movement is defined as evidence of fluorescence in 2 or more cells at 24 h and no movement is when fluorescence is in single cells. These were then presented as a percentage of the total. So by digitizing the representation of movement we were able to represent a linear relationship between movement and temperature. Representing movement in this way also enabled us to represent movement values on a logarithm scale necessary for a classic Arrhenius plot. The activation energy (Ea) was calculated by fitting the data to the Arrhenius equation:% movement = A exp(-Ea/RT); and the Ea for GFP and GFP-TGBp1 was approximately 38 kJ/mol and 29 kJ/mol. These low activation energies are comparable to the reported 30 kJ/mol calculated for temperature dependence of protein transport through the cytosol. Evidence that GFP movement across Pd requires slightly more energy than through the cytoplasm suggests there may be some resistance within the pore. The lower energy for GFP-TGBp1 suggests that movement is facilitated, which likely reflects Pd gating by TGBp1, enabling greater transfer between cells.Liarzi and Epel define a new coefficient of conductivity of Pd.⁴² This study also concluded that cell-to-cell transport of GFP in nontransgenic or transgenic N. benthamiana plants expressing the Tobacco mosaic virus (TMV) movement protein (MP) is temperature dependent. The method was to measure the exponential decay, which is a measure of the impedance to diffusion driven cell-to-cell movement of fluorescence. The exponential decay factor? was determined by calculating the ratio of GFP fluorescence in bombarded cell 0 and neighboring cells. This was presented as a measure of fluorescence transfer from cell 0 to cell 1 to cell 2. A coefficient for conductivity C(Pd), 1/? for GFP was reflective of diffusion. Interestingly the (TMV) MP did not increase conductivity of GFP between neighboring leaf epidermal cells indicating that movement was already maximal. Considering prior reports that the TMV MP shows preferential spread into mesophyll rather than epidermal tissues during virus infection, it is possible that preferential spread into mesophyll cells would prevent experimental efforts to achieve improved conductivity of GFP between epidermal cells.^27,28 In which case the absence of a trans effect of TMV MP on GFP conductivity in the epidermis may not be surprising. In fact, prior investigations of TMV MP gating activities were conducted in mesophyll cells.^29,30 The best explanation for the combined studies is that cytosolic GFP can diffuse across Pd , however viral proteins which gate Pd enable their own low energy transfer into neighboring cells without allowing other proteins to flood into neighboring cells. Therefore viral movement proteins, such as PVX TGBp1 and TMV MP, which gate Pd provide themselves with an energetic advantage for transport into neighboring cells which is essential for rapid dissemination of virus into further tissues.These studies provide an interesting contrast between PVX TGBp1 and TMV MP. Both proteins gate open Pd for virus cell-to-cell transport, but there seems to be differences in how these proteins function in epidermal cells. This is likely due to their different roles in promoting virus cell-to-cell movement. PVX TGBp1 protein is also a suppressor of RNA silencing. We recently proposed a model in which TGBp1 rapidly moves from cell-to-cell ahead of virus infection, to suppress the cell''s RNA degradation machinery, as a means to promote infection.³¹ The TMV MP on the other hand is reported to bind viral RNA for transfer into neighboring cells.^32,33 Therefore, the different observations of PVX TGBp1 and TMV MP transport between epidermal cells likely reflect their functional differences. Both proteins are required for virus cell-to-cell movement, but their exact roles in virus movement are not identical.As mentioned earlier, Pd permeability is downregulated during plant development. Research tracking GFP diffusion through Pd in embryonic cells, in young emerging leaves, and in fully expanded leaves showed that fluorescence is highly mobile between cells in young tissues but is restricted during maturation. Viral movement proteins such as Cucumber mosaic virus 3a, and PVX TGBp1 remain highly mobile in mature leaves because they gate open Pd under conditions that normally restrict movement of GFP.^34,35 Schoenknecht et al., undertook a straightforward investigation of leaf maturation describing Pd transport in relationship to leaf area expansion. The outcome of this study was evidence that GFP movement between cells declines as leaves expand.It is reasonable to consider that simultaneous changes in gene expression and physiology is reflected in a downward trend in Pd conductivity and an increased requirement for Pd gating to enable selected transport of macromolecules between cells. In Arabidopsis embryos there is an obvious transition between developmental stages which are also represented by a decline in the ability for GFP to diffuse across Pd.^36,37 A detailed analysis of Pd structure in source and sink tissues revealed that Pd are simple single channeled structures in sink tissues while source tissues contain predominantly “H” shaped branching Pd structures. The change in Pd structure has been correlated with changes in conductivity and is often correlated with changes in sink to source metabolism.^38,39 The sink-to-source transition in leaf development is typically monitored using phloem loading of carboxyfluorescein diacetate. Leaves where CF dye unloads are defined as sink leaves and leaves that were restricted in dye unloading were defined as source leaves. Then biolistic bombardment of GFP expressing plasmids to sink and source leaves revealed that GFP readily diffuses across Pd in sink leaves but is more often restricted in source leaves.^{26,34,40–42}Leaf development is typically defined as a transition from juvenile to adult which is represented by homeotic transformations as well as vegetative phase changes.^43,44 Source and sink regions of a leaf have been shown to correlate with changes in Pd structure and conductivity during leaf expansion. However, in our study we found that N. tabacum leaves identified as source during week 2 or 3 would continue to expand over an 8 week period to twice or three times the leaf area which provides a real indication that the source designation may not entirely reflect final leaf maturation or completion of leaf development.⁴⁵> For example, as cells transition from sink to source physiology it is suggested that the frequency of single channeled Pd declines while the frequency of branched Pd increases.³⁹ It is possible that even after leaves transition into photosynthetic sources that Pd architecture continues to change and there is a further decline in the proportion of single channel to branched channels. Therefore either the change in cell volume or Pd architecture or both can slow-down diffusion of GFP between cells.Researchers often point to the ER continuity between cells as a driving force for Pd formation and function. During cell division the cell wall is laid down and forms around the ER creating Pd channels.⁴⁶ However, it is also worth noting that the actin cytoskeleton is also present in Pd and is central to organ and reproductive development.^19,47 Actin and actin binding proteins are necessary for a number of plant processes determining the cell division plane, cell polarity, cell elongation, cytoplasmic streaming, transporting mRNAs and proteins, and defense.^48–51 Overexpression of ACT1 in Arabidopsis leaves can lead to changes in epidermal leaf shape and cause dwarfism in plants.⁵² Actin binding proteins are also necessary for organizing and remodeling the F-actin network which drives normal development of specific cell types and organs.⁵³ Actin filament bundling and remodeling are also seen in nonhost defense responses.⁵⁴ We do not know the effects of overexpressing certain actin homologues or actin remodeling on Pd formation or conductivity. Because the F-actin network is also central to Pd trafficking of proteins and macromolecules between cells it is worth considering F-actin as an early factor contributing to Pd formation which may be necessary to ensure cell-to-cell communication when cell polarity and elongation as well as defense machinery are being established.In summary, the novel quantitative tools developed for measuring protein movement across Pd reveal the temperature dependence of protein trafficking. Both the use of Arrhenius equation and C(Pd) provide new opportunities to measure the energy requirements for protein transport. These tools will enable researchers to quantify effects of environmental and developmental conditions on Pd conductivity, as well as comparing differences in Pd conductivity between plant species or induced by genetic mutations.     

Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states

Plant cells rely on plasmodesmata for intercellular transport of small signaling molecules as well as larger informational macromolecules such as proteins. A green fluorescent protein (GFP) reporter and low-pressure microprojectile bombardment were used to quantify the degree of symplastic continuity between cells of the leaf at different developmental stages and under different growth conditions. Plasmodesmata were observed to be closed to the transport of GFP or dilated to allow the traffic of GFP. In sink leaves, between 34% and 67% of the cells transport GFP (27 kD), and between 30% and 46% of the cells transport double GFP (54 kD). In leaves in transition transport was reduced; between 21% and 46% and between 2% and 9% of cells transport single and double GFP, respectively. Thus, leaf age dramatically affects the ability of cells to exchange proteins nonselectively. Further, the number of cells allowing GFP or double GFP movement was sensitive to growth conditions because greenhouse-grown plants exhibited higher diffusion rates than culture-grown plants. These studies reveal that leaf cell plasmodesmata are dynamic and do not have a set size exclusion limit. We also examined targeted movement of the movement protein of tobacco mosaic virus fused to GFP, P30::GFP. This 58-kD fusion protein localizes to plasmodesmata, consistently transits from up to 78% of transfected cells, and was not sensitive to developmental age or growth conditions. The relative number of cells containing dilated plasmodesmata varies between different species of tobacco, with Nicotiana clevelandii exhibiting greater diffusion of proteins than Nicotiana tabacum.     

Transient coexpression of individual genes encoded by the triple gene block of potato mop-top virus reveals requirements for TGBp1 trafficking

TGBp1, TGBp2, and TGBp3, three plant virus movement proteins encoded by the "triple gene block" (TGB), may act in concert to facilitate cell-to-cell transport of viral RNA genomes. Transient expression of Potato mop-top virus (genus Pomovirus) movement proteins was used as a model to reconstruct interactions between TGB proteins. In bombarded epidermal cells of Nicotiana benthamiana, green fluorescent protein (GFP)-TGBp1 was distributed uniformly. However, in the presence of TGBp2 and TGBp3, GFP-TGBp1 was directed to intermediate bodies at the cell periphery, and to cell wall-embedded punctate bodies. Moreover, GFP-TGBp1 migrated into cells immediately adjacent to the bombarded cell. These data suggest that TGBp2 and TGBp3 mediate transport of GFP-TGBp1 to and through plasmodesmata. Mutagenesis of TGBp1 suggested that the NTPase and helicase activities of TGBp1 were not required for its transport to intermediate bodies directed by TGBp2 and TGBp3, but these activities were essential for the protein association with cell wall-embedded punctate bodies and translocation of TGBpl to neighboring cells. The C-terminal region of TGBp1 was critical for trafficking mediated by TGBp2 and TGBp3. Mutation analysis also suggested an involvement of the TGBp2 C-terminal region in interactions with TGBp1.     

Development of a quantitative tool for measuring changes in the coefficient of conductivity of plasmodesmata induced by developmental, biotic, and abiotic signals

Summary. The regulation of intercellular and interorgan communication is pivotal for cell fate decisions in plant development and probably plays a significant role in the systemic regulation of gene expression and in defense reactions against pathogens or other biotic and abiotic environmental factors. In plants, symplasmic cell-to-cell communication is provided by plasmodesmata (Pd), coaxial membranous tunnels that span cell walls interconnecting adjacent cytoplasms. Macromolecules, proteins, and RNA may be transported through Pd by passive diffusion or by a facilitated mechanism. A quantitative tool was developed to measure the coefficient of conductivity, C(Pd), for diffusion-driven transport via Pd and to assess changes in the coefficient induced by developmental, biotic and abiotic signals. ^GFPC(Pd), the coefficient of conductivity for cell-to-cell spread of green-fluorescent protein (GFP), a protein with a Stokes radius of 2.82 nm, was determined in epidermal cells of sink and source leaves of wild-type and transgenic Nicotiana benthamiana plants expressing the movement protein of tobacco mosaic virus (MP^TMV) incubated both in dark and light and at 16 and 25°C. Under all conditions, Pd in source leaves conducted macromolecules, with ^GFPC(Pd)_sink > ^GFPC(Pd)_source. Light down-regulated ^GFPC(Pd) (all conditions); down-regulation was stronger for sink cells. The effect of MP^TMV on ^GFPC(Pd) between epidermal cells was dependent on temperature and leaf development; at 16°C, MP^TMV down-regulated ^GFPC(Pd) only in source leaves, while at 25°C, MP^TMV had no significant effect. This quantitative tool should be useful for investigating differences in Pd conductivity that are induced by mutations or silencing. Correspondence and reprints: Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Present address: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.     

A dysfunctional movement protein of tobacco mosaic virus interferes with targeting of wild-type movement protein to microtubules.

The Tobacco mosaic virus (TMV) movement protein (MPTMV) mediates cell-to-cell viral trafficking by altering properties of the plasmodesmata (Pd) in infected cells. During the infection cycle, MPTMV becomes transiently associated with endomembranes, microfilaments, and microtubules (MT). It has been shown that the cell-to-cell spread of TMV is reduced in plants expressing the dysfunctional MP mutant MPNT-1. To expand our understanding of the MP function, we analyzed events occurring during the intracellular and intercellular targeting of MPTMV and MPNT-1 when expressed as a fusion protein to green fluorescent protein (GFP), either by biolistic bombardment in a viral-free system or from a recombinant virus. The accumulation of MPTMV:GFP, when expressed in a viral-free system, is similar to MPTMV:GFP in TMV-infected tissues. Pd localization and cell-to-cell spread are late events, occurring only after accumulation of MP:GFP in aggregate bodies and on MT in the target cell. MPNT-1:GFP localizes to MT but does not target to Pd nor does it move cell to cell. The spread of transiently expressed MPTMV:GFP in leaves of transgenic plants that produce MPNT-1 is reduced, and targeting of the MPTMV:GFP to the cytoskeleton is inhibited. Although MPTMV:GFP targets to the Pd in these plants, it is partially impaired for movement. It has been suggested that MPNT-1 interferes with host-dependent processes that occur during the intracellular targeting program that makes MP movement competent.     

Mutations in the central domain of potato virus X TGBp2 eliminate granular vesicles and virus cell-to-cell trafficking

Most RNA viruses remodel the endomembrane network to promote virus replication, maturation, or egress. Rearrangement of cellular membranes is a crucial component of viral pathogenesis. The PVX TGBp2 protein induces vesicles of the granular type to bud from the endoplasmic reticulum network. Green fluorescent protein (GFP) was fused to the PVX TGBp2 coding sequence and inserted into the viral genome and into pRTL2 plasmids to study protein subcellular targeting in the presence and absence of virus infection. Mutations were introduced into the central domain of TGBp2, which contains a stretch of conserved amino acids. Deletion of a 10-amino-acid segment (m2 mutation) overlapping the segment of conserved residues eliminated the granular vesicle and inhibited virus movement. GFP-TGBp2m2 proteins accumulated in enlarged vesicles. Substitution of individual conserved residues in the same region similarly inhibited virus movement and caused the mutant GFP-TGBp2 fusion proteins to accumulate in enlarged vesicles. These results identify a novel element in the PVX TGBp2 protein which determines vesicle morphology. In addition, the data indicate that vesicles of the granular type induced by TGBp2 are necessary for PVX plasmodesmata transport.     

Nonspecific intercellular protein trafficking probed by green-fluorescent protein in plants

Summary Plasmodesmata mediate intercellular transport of proteins, nucleic acids, and small molecules in plants. We show that transiently produced green-fluorescent protein (GFP) trafficked intercellularly in the epidermis of sink leaves, but not of source leaves, in tobacco and cucumber. In contrast, the protein did not traffic in either sink or source leaves of tomato. On the other hand, the protein spread extensively from cell to cell in the epidermis of all leaves and stems ofArabidopsis thaliana as well as in young hypocotyls and cotyledons of tomato and cucumber. GFP could traffic from epidermis to ground tissues in hypocotyls but not in cotyledons of cucumber. GFP fused to a number of mutant forms of the cucumber mosaic virus 3a movement protein (CMV 3a MP) failed to traffic from cell to cell, suggesting that GFP does not have a specific motif for plasmodesmal trafficking. Our data, together with previous findings, indicate that plasmodesmata can mediate both specific and nonspecific intercellular trafficking of proteins. Furthermore, our data suggest that nonspecific protein trafficking is controlled by species-, development-, organ-, and tissue-specific factors. Since GFP can readily traffic from cell to cell, it raises the questions of how metabolites are compartmentalized intercellularly in a plant and of whether some endogenous plant proteins traffic nonspecifically from cell to cell to perform physiological functions yet to be elucidated.Abbreviations CMV cucumber mosaic virus - GFP green-fluorescent protein - MP movement protein - SEL size exclusion limit     

Respiratory energy demand for protein turnover and ion transport in wheat leaves upon water deficit

The effect of water stress on the respiratory energy demand for the main biosynthetic and transport processes was estimated in the leaves of spring wheat ( Triticum aestivum L. cv. San Pastore) acclimated and non-acclimated to drought. ATP-consuming processes were assessed from the effects of selective inhibitors of RNA synthesis, protein synthesis and proteolysis, Ca²⁺-ATPase and P-type ATPases on respiration. The proportions of energy consumed by these processes were compared with the theoretical ATP production calculated from the rate of oxygen consumption measured manometrically. Respiratory energy production increased significantly in both acclimated leaves and in leaves stressed by drought. In the fully grown wheat leaves, Ca²⁺-dependent reactions and protein turnover consumed about 37% and 34% of the total respiratory energy, respectively. The costs of ion transport constituted another 15% of the total ATP production. Both acclimation and drought stress in non-acclimated leaves resulted in a decrease of leaf sensitivity towards inhibitors of RNA and protein syntheses as well as a decrease in Ca²⁺-mediated processes; but also in an increase of leaf sensitivity towards inhibitors of proteolysis and ouabain-sensitive ATPase in non-acclimated plants. This indicates a shift in ATP input into the energy-requiring processes towards greater expenses for ion transport upon water deficit. However, in acclimated leaves under drought stress, distribution of respiratory energy became almost the same as in control plants.     

Dynamic changes in the frequency and architecture of plasmodesmata during the sink-source transition in tobacco leaves

Summary The sink-source transition in tobacco leaves was studied noninvasively using transgenic plants expressing the green-fluorescent protein (GFP) under control of theArabidopsis thaliana SUC2 promoter, and also by imaging transgenic plants that constitutively expressed a tobacco mosaic virus movement protein (MP) fused to GFP (MP-GFP). The sink-source transition was measured on intact leaves and progressed basipetally at rates of up to 600 m/h. The transition was most rapid on the largest sink leaves. However, leaf size was a poor indicator of the current position of the sink-source transition. A quantitative study of plasmodesmatal frequencies revealed the loss of enormous numbers of simple plasmodemata during the sink-source transition. In contrast, branched plasmodesmata increased in frequency during the sink-source transition, particularly between periclinal cell walls of the spongy mesophyll. The progression of plasmodesmal branching, as mapped by the labelling of plasmodesmata with MP-GFP fusion, occurred asynchronously in different cell layers, commencing in trichomes and appearing lastly in periclinal cell walls of the palisade layer. It appears that dividing cells retain simple plasmodesmata for longer periods than nondividing cells. The rapid conversion of simple to branched plasmodesmata is discussed in relation to the capacity for macromolecular trafficking in developing leaf tissues.     

Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana

Plant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. Functional entry sites into the leaf vascular system for rapid systemic infection have not been determined for any plant/virus system. Tobacco mosaic virus (TMV) entry into minor, major and transport veins from non-vascular cells of Nicotiana benthamiana in source tissue and its exit from veins in sink tissue was studied using a modified virus expressing green fluorescent protein (GFP). Using a surgical procedure that isolated specific leaf and stem tissues from complicating vascular tissues, we determined that TMV could enter minor, major or transport veins directly from non-vascular cells to produce a systemic infection. TMV first accumulated in abaxial or external phloem-associated cells in major veins and petioles of the inoculated leaf and stems below the inoculated leaf. It also initially accumulated exclusively in internal or adaxial phloem-associated cells in stems above the inoculated leaf and petioles or major veins of sink leaves. This work shows the functional equivalence of vein classes in source leaves for entry of TMV, and the lack of equivalence of vein classes in sink leaves for exit of TMV. Thus, the specialization of major veins for transport rather than loading of photoassimilates in source tissue does not preclude virus entry. During transport, the virus initially accumulates in specific vascular-associated cells, indicating that virus accumulation in this tissue is highly regulated. These findings have important implications for studies on the identification of symplasmic domains and host macromolecule vascular transport.     

Subcellular localization determines the availability of non-targeted proteins to plasmodesmatal transport

BACKGROUND: Individual plant cells are encased in a cell wall. To enable cell-to-cell communication, plants have evolved channels, termed plasmodesmata, to span thick walls and interconnect the cytoplasm between adjacent cells. How macromolecules pass through these channels is now beginning to be understood. RESULTS: Using two green fluorescent protein (GFP) reporters and a non-invasive transfection system, we assayed for intercellular macromolecular traffic in leaf epidermal cells. Plasmodesmata were found in different states of dilation. We could distinguish two forms of protein movement across plasmodesmata, non-targeted and targeted. Although leaves have generally been considered closed to non-specific transport of macromolecules, we found that 23% of the cells had plasmodesmatal channels in a dilated state, allowing GFP that was not targeted to plasmodesmata to move into neighboring cells. GFP fusions that were targeted to the cytoskeleton or to the endoplasmic reticulum did not move between cells, whereas those that were localized to the cytoplasm or nucleus diffused to neighboring cells in a size-dependent manner. Superimposed upon this non-specific exchange, proteins that were targeted to the plasmodesmata could transit efficiently between 62% of transfected cells. CONCLUSIONS: A significant population of leaf cells contain plasmodesmata in a dilated state, allowing macromolecular transport between cells. Protein movement potential is regulated by subcellular address and size. These parameters of protein movement illustrate how gradients of signaling macromolecules could be formed and regulated, and suggest that non-cell-autonomous development in plants may be more significant than previously assumed.     

Intramolecular complementing mutations in tobacco mosaic virus movement protein confirm a role for microtubule association in viral RNA transport

The movement protein (MP) of Tobacco mosaic virus (TMV) facilitates the cell-to-cell transport of the viral RNA genome through plasmodesmata (Pd). A previous report described the functional reversion of a dysfunctional mutation in MP (Pro81Ser) by two additional amino acid substitution mutations (Thr104Ile and Arg167Lys). To further explore the mechanism underlying this intramolecular complementation event, the mutations were introduced into a virus derivative expressing the MP as a fusion to green fluorescent protein (GFP). Microscopic analysis of infected protoplasts and of infection sites in leaves of MP-transgenic Nicotiana benthamiana indicates that MP(P81S)-GFP and MP(P81S;T104I;R167K)-GFP differ in subcellular distribution. MP(P81S)-GFP lacks specific sites of accumulation in protoplasts and, in epidermal cells, exclusively localizes to Pd. MP(P81S;T104I;R167K)-GFP, in contrast, in addition localizes to inclusion bodies and microtubules and thus exhibits a subcellular localization pattern that is similar, if not identical, to the pattern reported for wild-type MP-GFP. Since accumulation of MP to inclusion bodies is not required for function, these observations confirm a role for microtubules in TMV RNA cell-to-cell transport.     

Gene C2 of the monopartite geminivirus tomato yellow leaf curl virus-China encodes a pathogenicity determinant that is localized in the nucleus

Expression of the Tomato yellow leaf curl virus-China (TYLCV-C) C2 protein and green fluorescent protein (GFP) fused to the C2 protein (C2-GFP) in Nicotiana benthamiana from a Potato virus X (PVX) vector induced necrotic ringspots on inoculated leaves as well as necrotic vein banding and severe necrosis on systemically infected leaves. The localization of GFP fluorescence in plant cells infected with PVX/C2-GFP and in insect cells transfected with Baculovirus expressing C2-GFP indicates that the TYLCV-C C2 protein is capable of shuttling GFP into plant and insect cell nuclei. Our data demonstrate that the TYLCV-C C2 protein may contribute to viral pathogenicity in planta and is nuclear localized.     

Analysis of protein transport in the Brassica oleracea vasculature reveals protein-specific destinations

We investigated the vascular transport properties of exogenously applied proteins to Brassica oleracea plants and compared their delivery to various aerial parts of the plant with carboxy fluorescein (CF) dye. We identified unique properties for each protein. Alexafluor-tagged bovine serum albumin (Alexa-BSA) and Alexafluor-tagged Histone H1 (Alexa-Histone) moved slower than CF dye throughout the plant. Interestingly, Alexa-Histone was retained in the phloem and phloem parenchyma while Alexa-BSA moved into the apoplast. One possibility is that Alexa-Histone sufficiently resembles plant endogenous proteins and is retained in the vascular stream, while Alexa-BSA is exported from the cell as a foreign protein. Both proteins diffuse from the leaf veins into the leaf lamina. Alexa-BSA accumulated in the leaf epidermis while Alexa-Histone accumulated mainly in the mesophyll layers. Fluorescein-tagged hepatitis C virus core protein (fluorescein-HCV) was also delivered to B. oleracea plants and is larger than Alexa-BSA. This protein moves more rapidly than BSA through the plant and was restricted to the leaf veins. Fluorescein-HCV failed to unload to the leaf lamina. These combined data suggest that there is not a single default pathway for the vascular transfer of exogenous proteins in B. oleracea plants. Specific protein properties appear to determine their destination and transport properties within the phloem.     

Xylem structure and connectivity in grapevine (Vitis vinifera) shoots provides a passive mechanism for the spread of bacteria in grape plants

BACKGROUND AND AIMS: Bacterial leaf scorch occurring in a number of economically important plants is caused by the xylem-limited bacterium Xylella fastidiosa (Xf). In grapevine, Xf systemic infection causes Pierce's disease and is lethal. Traditional dogma is that Xf movement between vessels requires the digestion of inter-vessel pit membranes. However, Yersinia enterocolitica (Ye) (a bacterium found in animals) and fluorescent beads moved rapidly within grapevine xylem from stem into leaf lamina, suggesting open conduits consisting of long, branched xylem vessels for passive movement. This study builds on and expands previous observations on the nature of these conduits and how they affect Xf movement. METHODS: Air, latex paint and green fluorescence protein (GFP)-Xf were loaded into leaves and followed to confirm and identify these conduits. Leaf xylem anatomy was studied to determine the basis for the free and sometimes restricted movement of Ye, beads, air, paint and GFP-Xf into the lamina. KEY RESULTS: Reverse loading experiments demonstrated that long, branched xylem vessels occurred exclusively in primary xylem. They were observed in the stem for three internodes before diverging into mature leaves. However, this stem-leaf connection was an age-dependent character and was absent for the first 10-12 leaves basal to the apical meristem. Free movement in leaf blade xylem was cell-type specific with vessels facilitating movement in the body of the blade and tracheids near the leaf margin. Air, latex paint and GFP-Xf all moved about 50-60% of the leaf length. GFP-Xf was never observed close to the leaf margin. CONCLUSIONS: The open vessels of the primary xylem offered unimpeded long distance pathways bridging stem to leaves, possibly facilitating the spread of bacterial pathogens in planta. GFP-Xf never reached the leaf margins where scorching appeared, suggesting a signal targeting specific cells or a toxic build-up at hydathodes.     

Cell-to-cell trafficking of cucumber mosaic virus movement protein:green fluorescent protein fusion produced by biolistic gene bombardment in tobacco

Previous micro-injection studies showed that some recombinant viral movement proteins and plant proteins produced in and purified from Escherichia coli could traffic from cell to cell. However, the relevance of these findings obtained by micro-injecting proteins produced in E. coli to the real functions of these proteins when produced in planta has been questioned. In this study, specific gene constructs were delivered by biolistic bombardment into tobacco (Nicotiana tabacum var Samsun) leaf epidermis for in planta production of the green fluorescent protein (GFP) and various fusions between the cucumber mosaic virus 3a movement protein (3a MP) and GFP. Free GFP remained in cells producing it. In contrast, 3a MP:GFP fusion protein moved from approximately half of the cells producing it into neighboring cells. The movement also occurred at 4°C. A mutant 3a MP:GFP was incapable of cell-to-cell movement in all cases. A 3a MP:GUS fusion protein produced in this manner also moved from cell to cell. Our data provide direct evidence that specific viral proteins produced in planta can be transported between cells. Furthermore, our data suggest that the CMV 3a MP contains a signal for transport. Our approach is simple and efficient and has many potential applications in studying plasmodesma-mediated macromolecular transport.     

Developmental Regulation of Intercellular Protein Trafficking through Plasmodesmata in Tobacco Leaf Epidermis

Plasmodesmata mediate direct cell-to-cell communication in plants. One of their significant features is that primary plasmodesmata formed at the time of cytokinesis often undergo structural modifications, by the de novo addition of cytoplasmic strands across cell walls, to become complex secondary plasmodesmata during plant development. Whether such modifications allow plasmodesmata to gain special transport functions has been an outstanding issue in plant biology. Here we present data showing that the cucumber mosaic virus 3a movement protein (MP):green fluorescent protein (GFP) fusion was not targeted to primary plasmodesmata in the epidermis of young or mature leaves in transgenic tobacco (Nicotiana tabacum) plants constitutively expressing the 3a:GFP fusion gene. Furthermore, the cucumber mosaic virus 3a MP:GFP fusion protein produced in planta by biolistic bombardment of the 3a:GFP fusion gene did not traffic between cells interconnected by primary plasmodesmata in the epidermis of a young leaf. In contrast, the 3a MP:GFP was targeted to complex secondary plasmodesmata and trafficked from cell to cell when a leaf reached a certain developmental stage. These data provide the first experimental evidence, to our knowledge, that primary and complex secondary plasmodesmata have different protein-trafficking functions and suggest that complex secondary plasmodesmata may be formed to traffic specific macromolecules that are important for certain stages of leaf development.     

Spatial and temporal patterns of green fluorescent protein (GFP) fluorescence during leaf canopy development in transgenic oilseed rape,<Emphasis Type="Italic"> Brassica napus</Emphasis> L.

The green fluorescent protein (GFP) holds promise as a field-level transgene marker. One obstacle to the use of GFP is fluorescence variability observed within leaf canopies. In growth chamber and field experiments, GFP fluorescence in transgenic oilseed rape (Brassica napus) was shown to be variable at each leaf position over time and among different leaves on the same plant. A leaf had its highest GFP fluorescence after emergence and, subsequently, its fluorescence intensity decreased. GFP fluorescence intensity was directly correlated with the concentration of soluble protein. The concentration of the genetically linked recombinant Bacillus thuringiensis (Bt) cry1Ac endotoxin protein also was examined, and GFP fluorescence was positively correlated with Bt throughout development. The results show that GFP can be used as an accurate transgene marker but that aspects of plant developmental should be taken into account when interpreting fluorescence measurements.Communicated by M.C. Jordan     

Tobacco plants respond to the constitutive expression of the tospovirus movement protein NS(M) with a heat-reversible sealing of plasmodesmata that impairs development

Viral infection often results in typical symptoms, the biological background of which has remained elusive. We show that constitutive expression of the NSM viral movement protein (MP) of tomato spotted wilt virus in Nicotiana tabacum is sufficient to induce severe, infection-like symptoms, including pronounced deficiencies in root and shoot development. Leaves failed to expand and were arranged in a rosette due to the absence of internode elongation. Following the sink-source transition they accumulated excessive amounts of starch and developed fusing chlorotic patches in the mesophyll, resembling virus-induced chlorotic lesions. Eventually, the leaves became entirely white and brittle. With a combination of techniques, including photosystem II quantum-yield measurements, iontophoresis of symplasmic tracers, bombardment with pPVX.GFP and double immunolabelling it was shown that these symptoms correlated with the obstruction of NSM-targeted mesophyll plasmodesmata (Pd) in source tissues by depositions of 1,3-beta-D-glucan (GLU) or callose. Temperature-shift treatments (TST; 22-->32 degrees C), known to abolish chlorotic local lesions, also abolished the chlorotic 'superlesions' of transgenic plants and rescued plant development, by restoring the transport capacity of Pd through the action of 1,3-beta-D-glucanase (GLU-h) or callase. Return of these elongated, TST-recovered plants to 22 degrees C reintroduced superlesions and arrested shoot elongation, resulting in the formation of a rosette of clustered leaves at the shoot tip. Collectively, this indicates that the symptoms of NSM plants are self-inflicted and due to a basal defence response that counteracts prolonged interference of the MP with Pd functioning. This type of defence may also play a role in the formation of symptoms during viral infection.