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Targeting of TMV movement protein to plasmodesmata requires the actin/ER network: evidence from FRAP.

Fluorescence recovery after photobleaching (FRAP) was used to study the mechanism by which fluorescent-protein-tagged movement protein (MP) of tobacco mosaic virus (TMV) is targeted to plasmodesmata (PD). The data show that fluorescence recovery in PD at the leading edge of an infection requires elements of the cortical actin/endoplasmic reticulum (ER) network and can occur in the absence of an intact microtubule (MT) cytoskeleton. Inhibitors of the actin cytoskeleton (latrunculin and cytochalasin) significantly inhibited MP targeting, while MT inhibitors (colchicine and oryzalin) did not. Application of sodium azide to infected cells implicated an active component of MP transfer to PD. Treatment of cells with Brefeldin A (BFA) at a concentration that caused reabsorption of the Golgi bodies into the ER (precluding secretion of viral MP) had no effect on MP targeting, while disruption of the cortical ER with higher concentrations of BFA caused significant inhibition. Our results support a model of TMV MP function in which targeting of MP to PD during infection is mediated by the actin/ER network.

Actins↗

The product of maize streak virus ORF V1 is associated with secondary plasmodesmata and is first detected with the onset of viral lesions.

We have used a polyclonal antiserum derived from a bacterially expressed viral fusion protein to investigate the expression and subcellular localisation of the maize streak virus V1 product (PV1). Western blot analysis of agroinfected tissue showed that PV1 was detectable from 10 days postinoculation, coinciding with the first appearance of chlorotic viral lesions. The viral protein was only detectable in cell wall fractions of plant protein extracts. PV1 migrated with an apparent size of 14 kDa on SDS-PAGE, larger than the 10.9 kDa predicted from the amino acid sequence and therefore suggestive of posttranslational modification. Immunogold labelling located PV1 to the cell walls within lesion tissue and demonstrated a close association between the viral protein and secondary plasmodesmata. These results are consistent with the V1 product of MSV playing a role in the cell-to-cell movement of the virus in infected plants.

Base Sequence↗

Cell wall synthesis in cotton roots after infection with Fusarium oxysporum. The deposition of callose, arabinogalactans, xyloglucans, and pectic components into walls, wall appositions, cell plates and plasmodesmata.

Fusarium oxysporum f. sp. vasinfectum penetration hyphae infect living cells in the meristematic zone of cotton (Gossypium barbadense L.) roots. We characterized wall modifications induced by the fungus during infection of the protodermis using antibodies against callose, arabinogalactan-proteins, xyloglucan, pectin, polygalacturonic acid and rhamnogalacturonan I in high-pressure frozen, freeze-substituted root tissue. Using quantitative immunogold labelling we compared the cell walls before and after hyphal contact, cell plates with plasmodesmata during cytokinesis, and wall appositions induced by fungal contact. In the already-existing wall, fungal contact induced only minor modifications such as an increase of xyloglucan epitopes. Wall appositions mostly exhibited epitopes similar to the cell plate except that wall appositions had a much higher callose content. This study shows that wall appositions induced by Fusarium oxysporum hyphae are the result of normal cell wall synthesis and the addition of large amounts of callose. The appositions do not stop fungal growth.

Cell Wall↗

Plasmodesmata. Gateways for rapid information transfer.

Viruses spread their genomes throughout infected plants by exploiting plasmodesmata, the cytoplasmic bridges that wire plant cells into a three-dimensional network and rapidly transport a variety of molecules.

Biological Transport↗

Plasmodesmata signaling: many roles, sophisticated statutes.

Recent advances have increased our understanding of plasmodesmata function, their architecture as it relates to signaling capacity, the temporal and spatial regulation of their permeability, and their roles in systemic transport of macromolecules, non-cell autonomous development, and, potentially, plant defense.

Cell Wall↗

Aluminum-induced 1-->3-beta-D-glucan inhibits cell-to-cell trafficking of molecules through plasmodesmata. A new mechanism of aluminum toxicity in plants.

Symplastic intercellular transport in plants is achieved by plasmodesmata (PD). These cytoplasmic channels are well known to interconnect plant cells to facilitate intercellular movement of water, nutrients, and signaling molecules including hormones. However, it is not known whether Al may affect this cell-to-cell transport process, which is a critical feature for roots as organs of nutrient/water uptake. We have microinjected the dye lucifer yellow carbohydrazide into peripheral root cells of an Al-sensitive wheat (Triticum aestivum cv Scout 66) either before or after Al treatment and followed the cell-to-cell dye-coupling through PD. Here we show that the Al-induced root growth inhibition is closely associated with the Al-induced blockage of cell-to-cell dye coupling. Immunofluorescence combined with immuno-electron microscopic techniques using monoclonal antibodies against 1-->3-beta-D-glucan (callose) revealed circumstantial evidence that Al-induced callose deposition at PD may responsible for this blockage of symplastic transport. Use of 2-deoxy-D-glucose, a callose synthesis inhibitor, allowed us to demonstrate that a reduction in callose particles correlated well with the improved dye-coupling and reduced root growth inhibition. While assessing the tissue specificity of this Al effect, comparable responses were obtained from the dye-coupling pattern in tobacco (Nicotiana tabacum) mesophyll cells. Analyses of the Al-induced expression of PD-associated proteins, such as calreticulin and unconventional myosin VIII, showed enhanced fluorescence and co-localizations with callose deposits. These results suggest that Al-signal mediated localized alterations to calcium homeostasis may drive callose formation and PD closure. Our data demonstrate that extracellular Al-induced callose deposition at PD could effectively block symplastic transport and communication in higher plants.

Aluminum↗

Cell-to-Cell Trafficking of Macromolecules through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein.

Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluorescently labeled 35-kD movement protein of red clover necrotic mosaic virus (RCNMV) trafficked rapidly from cell to cell when microinjected into cowpea leaf mesophyll cells. Furthermore, this protein potentiated rapid cell-to-cell trafficking of RCNMV RNA, but not DNA. Electron microscopic studies demonstrated that the 35-kD movement protein does not unfold the RCNMV RNA molecules. Thus, if unfolding of RNA is necessary for cell-to-cell trafficking, it may well involve participation of endogenous cellular factors. These findings support the hypothesis that trafficking of macromolecules is a normal plasmodesmal function, which has been usurped by plant viruses for their cell-to-cell spread.

Journal Article↗

Plasmodesmata between hyphal cells of Geotrichum candidum.

Electron-microscopic studies of three isolates of Geotrichum candidum from plants and one isolate from man showed the fine structure to be similar to other closely related, yeastlike fungi. Structures were observed that appeared similar to plasmodesmata present in higher plants.

Humans↗

Phloem loading in two Scrophulariaceae species. What can drive symplastic flow via plasmodesmata?

To determine the driving forces for symplastic sugar flux between mesophyll and phloem, gradients of sugar concentrations and osmotic pressure were studied in leaf tissues of two Scrophulariaceae species, Alonsoa meridionalis and Asarina barclaiana. A. meridionalis has a typical symplastic configuration of minor-vein phloem, i.e. intermediary companion cells with highly developed plasmodesmal connections to bundle-sheath cells. In A. barclaiana, two types of companion cells, modified intermediary cells and transfer cells, were found in minor-vein phloem, giving this species the potential to have a complex phloem-loading mode. We identified all phloem-transported carbohydrates in both species and analyzed the levels of carbohydrates in chloroplasts, vacuoles, and cytoplasm of mesophyll cells by nonaqueous fractionation. Osmotic pressure was measured in single epidermal and mesophyll cells and in whole leaves and compared with calculated values for phloem sap. In A. meridionalis, a 2-fold concentration gradient for sucrose between mesophyll and phloem was found. In A. barclaiana, the major transported carbohydrates, sucrose and antirrhinoside, were present in the phloem in 22- and 6-fold higher concentrations, respectively, than in the cytoplasm of mesophyll cells. The data show that diffusion of sugars along their concentration gradients is unlikely to be the major mechanism for symplastic phloem loading if this were to occur in these species. We conclude that in both A. meridionalis and A. barclaiana, apoplastic phloem loading is an indispensable mechanism and that symplastic entrance of solutes into the phloem may occur by mass flow. The conditions favoring symplastic mass flow into the phloem are discussed.

Biological Transport↗