Inhibition of tobacco mosaic virus replication in lateral roots is dependent on an activated meristem-derived signal.

Viral invasion of the root system of Nicotiana benthamiana was studied noninvasively with a tobacco mosaic virus (TMV) vector expressing the green-fluorescent protein (GFP). Lateral root primordia, which developed from the pericycle of primary roots, became heavily infected as they emerged from the root cortex. However, following emergence, a progressive wave of viral inhibition occurred that originated in the lateral-root meristem and progressed towards its base. Excision of source and sink tissues suggested that the inhibition of virus replication was brought about by the basipetal movement of a root meristem signal. When infected plants were inoculated with tobacco rattle virus (TRV) expressing the red-fluorescent protein, DsRed, TRV entered the lateral roots and suppressed the host response, leading to a reestablishment of TMV infection in lateral roots. By infecting GFP-expressing transgenic plants with TMV carrying the complementary GFP sequence it was possible to silence the host GFP, leading to the complete loss of fluorescence in lateral roots. The data suggest that viral inhibition in lateral roots occurs by a gene-silencing-like mechanism that is dependent on the activation of a lateral-root meristem.

Splicing signals and factors in plant intron removal.

Constitutive splicing of the potato invertase mini-exon 2 (9 nt long) requires a branchpoint sequence positioned around 50 nt upstream of the 5' splice site of the adjacent intron and a U(11) element found just downstream of the branchpoint in the upstream intron [Simpson, Hedley, Watters, Clark, McQuade, Machray and Brown (2000) RNA 6, 422-433]. The sensitivity of this in vivo plant splicing system has been used to demonstrate exon scanning in plants, and to characterize plant intronic elements, such as branchpoint and poly-pyrimidine tract sequences. Plant introns differ from their vertebrate and yeast counterparts in being UA- or U-rich (up to 85% UA). One of the key differences in splicing between plants and other eukaryotes lies in early intron recognition, which is thought to be mediated by UA-binding proteins. We are adopting three approaches to studying the RNA-protein interactions in plant splicing. First, overexpression of plant splicing factors and, in particular, UA-binding proteins, in conjunction with a range of mini-exon mutants. Secondly, the sequences of around 65% of vertebrate and yeast splicing factors have high-quality matches to Arabidopsis proteins, opening the door to identification and analysis of gene knockouts. Finally, to discover plant-specific proteins involved in splicing and in, for example, rRNA or small nuclear RNA processing, green fluorescent protein-cDNA fusion libraries in viral vectors are being screened.

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

The sink-source transition in tobacco leaves was studied noninvasively using transgenic plants expressing the green-fluorescent protein (GFP) under control of the Arabidopsis 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 microns/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.

Tuberization in potato involves a switch from apoplastic to symplastic phloem unloading.

Phloem unloading was studied in potato plants in real time during the early stages of tuberization using carboxyfluorescein (CF) as a phloem-mobile tracer, and the unloading pattern was compared with autoradiography of tubers that had transported (14)C assimilates. In stolons undergoing extension growth, apoplastic phloem unloading predominated. However, during the first visible signs of tuberization, a transition occurred from apoplastic to symplastic transport, and both CF and (14)C assimilates subsequently followed identical patterns of phloem unloading. It is suggested that the switch to symplastic sucrose unloading may be responsible for the upregulation of several genes involved in sucrose metabolism. A detailed analysis of sugar levels and (14)C sugar partitioning in tuberizing stolons revealed a distinct difference between the apical region of the tuber and the subapical region. Analysis of invertase activity in nontuberizing and tuberizing stolons revealed a marked decline in soluble invertase in the subapical region of swelling stolons, consistent with the switch from apoplastic to symplastic unloading. However, cell wall-bound invertase activity remained high in the apical 1 to 2 mm of tuberizing stolons. Histochemical analysis of potato lines transformed with the promoter of an apoplastic invertase gene (invGE) linked to a reporter gene also revealed discrete gene expression in the apical bud region. Evidence is presented that the apical and lateral tuber buds function as isolated domains with respect to sucrose unloading and metabolism.

Macromolecular trafficking between Nicotiana tabacum and the holoparasite Cuscuta reflexa.

Transgenic tobacco plants expressing green fluorescent protein (GFP) under the control of the companion cell-specific promoter, AtSUC2, were parasitized by the holoparasite Cuscuta reflexa (dodder). GFP, moving in the translocation stream of the host, was transferred to the Cuscuta phloem via the absorbing hyphae of the parasite. An identical pattern of transfer was observed for the phloem-mobile probe, carboxyfluorescein. Following uptake by the parasite, GFP was translocated and unloaded from the Cuscuta phloem in meristematic sink tissues. Contrary to published data, these observations suggest the presence of a functional symplastic pathway between Cuscuta and its hosts, and demonstrate a considerable capacity for macromolecular exchange between plant species.

Evidence for symplastic phloem unloading in sink leaves of barley.

The pathway of phloem unloading in sink barley (Hordeum vulgare) leaves was studied using a combination of electron microscopy, carboxyfluorescein transport, and systemic movement of barley stripe mosaic virus expressing the green fluorescent protein. Studies of plasmodesmatal frequencies between the phloem and mesophyll indicated a symplastic sieve element- (SE) unloading pathway involving thick-walled and thin-walled SEs. Phloem-translocated carboxyfluorescein was unloaded rapidly from major longitudinal veins and entered the mesophyll cells of sink leaves. Unloading was "patchy" along the length of a vein, indicating that sieve element unloading may be discontinuous along a single vascular bundle. This pattern was mirrored precisely by the unloading of barley stripe mosaic virus expressing the green fluorescent protein. Transverse veins were not utilized in the unloading process. The data collectively indicate a symplastic mechanism of SE unloading in the sink barley leaf.

Analysis of the N gene hypersensitive response induced by a fluorescently tagged tobacco mosaic virus.

The hypersensitive response (HR) triggered on Nicotiana edwardsonii by tobacco mosaic virus was studied using a modified viral genome that directed expression of the green fluorescent protein. Inoculated plants were initially incubated at 32 degrees C to inhibit the N gene-mediated HR. Transfer to 20 degrees C initiated the HR, and fluorescent infection foci were monitored for early HR-associated events. Membrane damage, which preceded visible cell collapse by more than 3 h, was accompanied by a transient restriction of the xylem within infection sites. Following cell collapse and the rapid desiccation of tissue undergoing the HR, isolated, infected cells were detected at the margin of necrotic lesions. These virus-infected cells were able to reinitiate infection on transfer to 32 degrees C, however, if maintained at 20 degrees C they eventually died. The results indicate that the tobacco mosaic virus-induced HR is a two-phase process with an early stage culminating in rapid cell collapse and tissue desiccation followed by a more extended period during which the remaining infected cells are eliminated.

Maize streak virus coat protein is karyophyllic and facilitates nuclear transport of viral DNA.

Transport of maize streak virus (MSV) DNA into the nucleus of host cells is essential for virus replication and the presence of virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus. To see if CP is imported into the nucleus in the absence of other viral gene products, the MSV CP gene was expressed in insect cells with a baculovirus vector system, and also in tobacco protoplasts with a cauliflower mosaic virus (CaMV) 35S promoter-driven transient gene expression vector. Immunofluorescent staining showed that the CP accumulated in the nuclei of both insect and tobacco cells. Mutagenesis of a potential nuclear localization signal in the CP resulted in cytoplasmic accumulation of the mutant protein. We have shown previously that the CP binds to single-stranded (ss) and double-stranded (ds) viral DNA. To investigate if CP might also be involved in viral DNA nuclear transport, Escherichia coli-expressed CP, together with TOTO-1-labeled viral ss or ds DNA, was microinjected into maize and tobacco epidermal cells. Both ss and ds DNA moved into the nucleus when co-injected with the CP but not with E. coli proteins alone. These results suggest that, in addition to entering the nucleus where it is required for encapsidation of the viral ss DNA, the MSV CP facilitates the rapid transport of viral (ss or ds) DNA into the nucleus.

Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves.

Leaves undergo a sink-source transition during which a physiological change occurs from carbon import to export. In sink leaves, biolistic bombardment of plasmids encoding GFP-fusion proteins demonstrated that proteins with an Mr up to 50 kDa could move freely through plasmodesmata. During the sink-source transition, the capacity to traffic proteins decreased substantially and was accompanied by a developmental switch from simple to branched forms of plasmodesmata. Inoculation of sink leaves with a movement protein-defective virus showed that virally expressed GFP, but not viral RNA, was capable of trafficking between sink cells during infection. Contrary to dogma that plasmodesmata have a size exclusion limit below 1 kDa, the data demonstrate that nonspecific "macromolecular trafficking" is a general feature of simple plasmodesmata in sink leaves.

En bloc optical sectioning of resin-embedded specimens using a confocal laser scanning microscope.

Reconstruction of 3D structures of specimens embedded for light or electron microscopy is usually achieved by cutting serial sections through the tissues, then assembling the images from each section to reconstruct the original structure or feature. This is both time-consuming and destructive, and may lead to areas of particular interest being missed. This paper describes a method of examining specimens which have been fixed in glutaraldehyde and embedded in epoxy resin, by utilising the autofluorescence preserved or enhanced by aldehyde fixation, and by using a confocal laser scanning microscope to section optically such specimens in the block down to a depth of about 200 &mgr;m. In this way, the accurate estimation of the depth of particular features could be used to facilitate subsequent sectioning at the light microscope or electron microscope level for more detailed studies, and 3D images of tissues/structures within the block could be easily prepared if required.

Intracellular location of two groundnut rosette umbravirus proteins delivered by PVX and TMV vectors.

The proteins encoded by open reading frames (ORF) 3 and 4 of groundnut rosette umbravirus (GRV) were expressed in Nicotiana benthamiana as fusions with green fluorescent protein (GFP) from modified potato virus X (PVX) and tobacco mosaic virus (TMV) vectors. Regardless of which plant virus vector was used, GFP fused to the ORF3 protein accumulated in large cytoplasmic inclusion bodies and in nucleoli, whereas GFP fused to the ORF4 protein was found in cell walls close to plasmodesmata. Cell-to-cell movement of PVX requires three proteins encoded by the triple gene block (TGB) and also the coat protein (CP). However, when GRV ORF4 was substituted for the PVX CP gene, the hybrid virus was able to move normally in inoculated leaves but not into noninoculated leaves. In contrast, when GRV ORF4 was substituted for the TGB, or for both the TGB and the CP gene, movement of the hybrid viruses was limited to a few epidermal cells neighboring the infection site. Thus, the GRV ORF4 protein can replace the movement proteins of PVX for some of their functions.

Characterization of cucumber mosaic virus. IV. Movement protein and coat protein are both essential for cell-to-cell movement of cucumber mosaic virus.

cDNA clones of cucumber mosaic virus (CMV) RNA 3 were modified to express the jellyfish green fluorescent protein (GFP) in place of the 3a movement protein (MP) or coat protein (CP), as fusions to the N (GFP-3a) or C (3a-GFP) terminus of the MP or from a separate open reading frame as part of tricistronic RNAs 3. CMV RNA transcripts containing the individual modified RNAs 3 were unable to infect either Nicotiana tabacum or Nicotiana benthamiana systemically. Infection, as measured by confocal microscopy of GFP fluorescence, generally was limited to one to three epidermal cells at each inoculation site. Limited cell-to-cell movement, but not systemic movement, could be detected by complementation involving expression of MP and CP from two different RNA 3 constructs, each also expressing GFP. Infection involving RNA 3 expressing the GFP-3a fusion showed bright granules of variable size distributed predominantly and nonuniformly throughout the cytoplasm and, to a lesser extent, associated with the cell wall in single fluorescent cells, while infections expressing the 3a-GFP fusion showed bright, punctate fluorescence associated only with the cell wall. Infected cells expressing either 3a-GFP or free GFP showed a halo of less bright, fluorescent, neighboring cells, indicating limited movement of GFP. The initially infected cells also allowed movement of 10-kDa fluorescent dextran to the neighboring halo cells, while infection did not spread, suggesting different requirements for movement of either MP or dextran versus RNA.

Phloem Unloading in Sink Leaves of Nicotiana benthamiana: Comparison of a Fluorescent Solute with a Fluorescent Virus.

Using noninvasive imaging techniques, we compared phloem unloading of the membrane-impermeant, fluorescent solute carboxyfluorescein (CF) with that of potato virus X expressing the gene for the green fluorescent protein. Although systemic virus transport took considerably longer to occur than did CF transport, unloading of both solute and virus occurred predominantly from the class III vein network, a highly branched veinal system found between class II veins. The minor veins (classes IV and V) played no role in solute or virus import but were shown to be functional in xylem transport at the time of import by labeling with Texas Red dextran. After virus exit from the class III phloem, the minor veins eventually became infected by cell-to-cell virus movement from the mesophyll. During the sink/source transition, phloem unloading of CF was inhibited from class III veins before the cessation of phloem import through them, suggesting a symplastic isolation of the phloem in class III veins before its involvement in export. The progression of the sink/source transition for carbon was unaffected by the presence of the virus in the sink leaf. However, the virus was unable to cross the sink/source boundary for carbon that was present at the time of viral entry, suggesting a limited capacity for cell-to-cell virus movement into the apical (source) region of the leaf. A functional model of the sink/source transition in Nicotiana benthamiana is presented. This model provides a framework for the analysis of solute and virus movement in leaves.

Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV).

Plasmodesmatal gating in epidermal cells of Nicotiana tabacum was examined in expanding infection sites of tobacco mosaic virus (TMV) expressing a fusion between the viral movement protein and the green fluorescent protein (MP-GFP). The infection sites were circular in profile and within 3 days post-inoculation had developed a brightly fluorescent leading edge, giving them a characteristic 'halo' shape. Co-localization of MP-GFP with callose demonstrated that nearly all epidermal cell plasmodesmata were targeted with MP-GFP. The fusion protein was located in the centre of the plasmodesmal pore, between paired callose platelets. Increase in plasmodesmatal size exclusion limit, as determined by the passage of microinjected 10 kDa Texas Red dextran, was restricted predominantly to cells within the fluorescent halo, and was virtually absent from cells in the centre of the expanding infection site. The plasmodesmata of these cells, however, remained fluorescently labelled with MP-GFP. Injections outside the fluorescent infection site failed to show movement of dextran, while dextran injected into cells at the leading edge moved inwards towards the centre of the lesion but not outwards into cells lacking GFP. Leaf incisions through cells ahead of the infection front halted the advance of the virus, indicating that virus replication was absent in non-fluorescent cells outside the infection site. The data provide the first demonstration that within an expanding infection site plasmodesmatal gating is under temporal control.

Induction of phloem unloading in Arabidopsis thaliana roots by the parasitic nematode Heterodera schachtii.

Phloem unloading of both the fluorescent probe carboxyfluorescein (CF) and 14C-labeled solutes was induced in Arabidopsis thaliana L. roots by the parasitic nematode Heterodera schachtii Schmidt. Confocal laser scanning microscopy demonstrated that anomalous unloading of CF from the sieve element companion cell complexes occurred specifically into the syncytium, the nematode-induced feeding structure located within the stele of the root. From this syncytial complex of modified root cells, both fluorescent and radioactive labels were withdrawn by feeding nematodes. Movement of CF was unidirectional from the phloem to the syncytium. A range of low-molecular-weight fluorescent probes (including CF) microinjected into the syncytium stayed in this structure, demonstrating that it is symplastically isolated from the surrounding root tissue. The mechanism of unloading in this host-pathogen relationship therefore appears to be apoplastic. Our results provide unequivocal evidence that sedentary cyst-forming nematodes have direct access to phloem-derived solutes.

Assembly and movement of a plant virus carrying a green fluorescent protein overcoat.

Potato virus X (PVX) is a filamentous plant virus infecting many members of the family Solanaceae. A modified form of PVX, PVX.GFP-CP which expressed a chimeric gene encoding a fusion between the 27-kDa Aequorea victoria green fluorescent protein and the amino terminus of the 25-kDa PVX coat protein, assembled into virions and moved both locally and systemically. The PVX.GFP-CP virions were over twice the diameter of wild-type PVX virions. Assembly of PVX.GFP-CP virions required the presence of free coat protein subunits in addition to the fusion protein subunits. PVX.GFP-CP virions accumulated as paracrystalline arrays in infected cells similar to those seen in cells infected with wild-type PVX The formation of virions carrying large superficial fusions illustrates a novel approach for production of high levels of foreign proteins in plants. Aggregates of PVX.GFP-CP particles were fluorescent, emitting green light when excited with ultraviolet light and could be imaged using confocal laser scanning microscopy. The detection of virus particles in infected tissue demonstrates the potential of fusions between the green fluorescent protein and virus coat protein for the non-invasive study of virus multiplication and spread.

Post-phloem transport: principles and problems.

The movement of assimilates from the sieve element/companion cell complex to sites of utilization has been examined in an extensive array of sinks possessing diverse anatomies. This work has been reviewed with respect to the pathways taken, the conductances and driving forces for movement along the pathways, and interaction between the apoplast and symplast. Most investigations to date have been concerned primarily with determining the pathway of assimilate movement. A symplastic pathway is followed in the great majority of cases studied. However, available methods are less suited for demonstrating apoplastic transport in those instances where it occurs. Far less information is available on quantitative aspects of post-phloem transport. Only a very limited number of observations are available on the diffusive or hydraulic conductances of the apoplast or symplast. In some cases, symplastic conductance appears to be enhanced by a larger-than-usual size exclusion limit for cell-to-cell transport. Measurements of the driving forces for post-phloem transport (i.e. gradients in concentration and/or pressure) are also very few in number nor, to date, are they always readily interpretable. Evaluation of solute movement is complicated by interactions between the apoplastic and symplastic pathways, including water relations effects and solute exchange. The presence of apoplastic domains or, simply, high resistance to movement in the apoplast, can lead to steep water relations gradients within sinks, with important implications for transport. To understand how import into sinks is controlled, many more quantitative measurements are needed. This will require considerable experimental ingenuity.

Signalling via plasmodesmata--the neglected pathway.

This review considers recent studies on the role of plasmodesmata in the conduction of small solutes and signalling molecules between plant cells. The substructure of plasmodesmata is described in relation to the potential pathways available for symplastic signalling between cells. At least two discrete pathways are available for transport through plasmodesmata, the cytoplasmic sleeve between the desmotubule and the plasmalemma, and the endoplasmic reticulum which connects contiguous cells via the central desmotubule. This latter pathway has been shown recently to function as a dynamic continuum for the movement of lipids and lipid-signalling molecules between plant cells. The role of plasmodesmata in the conduction of hormones and electrical signals is also considered, as is the potential for movement of macromolecular signalling molecules via the symplast. The factors which regulate plasmodesmatal conductance and the significance of symplast 'domains' are discussed in relation to the control of movement of signalling molecules in the symplast.