The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection - PubMed (original) (raw)

The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection

Ho-Jong Ju et al. Plant Physiol. 2005 Aug.

Abstract

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virus-infected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.

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Figures

Figure 1.

Figure 1.

Schematic representation of plasmids used in this study. The pPVX-GFP and pPVX-GFP:TGBp2 plasmids contain the entire PVX genome. The white boxes in these schematics represent the PVX ORFs or subcellular targeting signals, and the lines represent noncoding regions. The names for each PVX gene are indicated above the boxes. The GFP gene (gray box) or GFP:TGBp2-fused genes were inserted into the PVX genome. In the pPVX-GFP:TGBp2 plasmid, a large portion of the endogenous TGBp2 coding sequence was deleted from the PVX genome (indicated by a black box). Four pRTL2 plasmids expressed GFP alone (gray box) or fused to the 5′-end of the PVX TGBp1, TGBp2, or TGBp3 genes (white boxes). The pBIN-mGFP5-ER has sequences encoding ER-targeting and retention signals fused to the 5′- and 3′-ends of the GFP gene. The pRTL2-GFP:Talin and -DsRed:Talin plasmids have the coding sequence of the F-actin-binding domain of mouse Talin (McCann and Craig, 1997; Kost et al., 1998) for actin targeting of GFP or DsRed (hatched box). The pDsRed-ST plasmid has sequences encoding the Golgi-targeting signal of rat ST fused to DsRed (Dixit and Cyr, 2002). The DsRed-ST fusion is in the pCambia1300 vector (Dixit and Cyr, 2002). All pRTL2, pBIN, and pCambia1300 plasmids contain a CaMV 35S promoter (black spotted arrows).

Figure 2.

Figure 2.

Confocal images of protoplasts infected with PVX-GFP:TGBp2 and PVX-GFP. A to C, PVX-GFP:TGBp2-infected protoplasts show fluorescence in vesicles (A); vesicles and aggregates of vesicles (B); and vesicles, nucleus (n), and cytoplasm (C). D, A magnified portion of the protoplast presented in C. Vesicles appear along the plasma membrane and surrounding the nucleus. E and F, PVX-GFP:TGBp2 (E) and PVX-GFP-infected protoplast (F) show nuclear and cytoplasmic fluorescence. G to I, DAPI-stained protoplasts infected with PVX-GFP:TGBp2. G, Dividing protoplasts have two adjacent nuclei stained with DAPI. GFP fluorescent vesicles surround the nuclei in G and H. I, DAPI-stained cell shows GFP:TGBp2 in the nucleus and cytoplasm. Bars in each photograph represent 10 _μ_m. J, Thirty protoplasts were scored for the presence of fluorescence in vesicles, nucleus, and cytoplasm in PVX-GFP:TGBp2-infected protoplasts at 18, 24, 36, 48, and 72 hpi. K, Thirty protoplasts were scored for the presence of fluorescence in vesicles, nucleus, and cytoplasm in PVX-GFP-infected protoplasts at 18, 24, 36, 48, and 72 hpi. Bars represent the percentage of protoplasts having fluorescence in each subcellular compartment at each time point. Between 1% and 10% of protoplasts at each time point contained GFP in vesicles resembling those seen in the PVX-GFP:TGBp2-infected cells. Logistic regressions were conducted using data in J and K and reported in “Results.”

Figure 3.

Figure 3.

Confocal images of PVX-GFP and PVX-GFP:TGBp2-infected N. benthamiana leaf epidermal cells. A and B, PVX-GFP-infected cells at 2 and 6 dpi, respectively. Green fluorescence is present in the cytoplasm and nucleus. There were also perinuclear inclusion bodies that, on occasion, seemed to surround the nucleus. Since the perinuclear inclusions often overlap the nucleus, single arrowheads point to both structures in all images. C to G, PVX-GFP:TGBp2-infected cells at 2 or 4 dpi. Single arrowheads point to inclusion bodies and arrows point to vesicles. C, Image of a PVX-GFP:TGBp2 infection focus taken at low magnification at 4 dpi. D, Image of cells located at the front of an infection focus. Fluorescence was evident in the ER network, vesicles, and inclusion bodies. E and F, Single optical cross-sections through the middle of the cell located at the front of an infection focus. The vesicles appear as twin structures along the walls of opposing cells. G and H, Images of cells located in the center of an infection focus at 4 dpi. These images show fluorescence in a thick layer of cytoplasm around the cell, the ER, and vesicles. The nuclear fluorescence is either due to GFP:TGBp2 accumulating inside the nucleus, perinuclear X-bodies that overwhelm the nucleus (Kikumoto and Matsui, 1961; Kozar and Sheludko, 1969; Allison and Shalla, 1973), or both. Bars in all images, except C, represent 20 _μ_m; bar in C represents 200 _μ_m.

Figure 4.

Figure 4.

Fluorometric assays measuring GFP fluorescence in BY-2 protoplasts. The averages of three fluorometric values were plotted at each time point. A, PVX-GFP (black triangle) and PVX-GFP:TGBp2 (white circle) infected protoplasts. The average fluorometric values were plotted using linear regression. B, PVX-GFP (black triangles) and PVX-GFP:TGBp2 (white circles) infected protoplasts treated with cycloheximide at 24 hpt. The average fluorometric values were normalized to an average time 0 measurement and then plotted using linear regression. C, pRTL2-GFP (black diamonds) and pRTL2-GFP:TGBp2 (white squares) transfected protoplasts. The average fluorometric values were plotted and a best-fit curve was determined using polynomial regression. D, pRTL2-GFP (black diamonds) and pRTL2-GFP:TGBp2 (white squares) transfected protoplasts were treated with cycloheximide at 24 hpt. The average fluorometric values were normalized to an average time 0 measurement and then plotted using linear regression.

Figure 5.

Figure 5.

Confocal images showing subcellular accumulation of fluorescent proteins expressed transiently in BY-2 protoplasts and N. benthamiana leaves. Arrows point to aggregates and arrowheads point to vesicles in A to C. A, D, F, G, and I, Images of protoplasts. B, C, E, H, and J, Images of tobacco leaf epidermal cells. A to C, pRTL2-GFP:TGBp2-transfected protoplasts and cells. D and E, pRTL2-GFP:TGBp3-transfected protoplasts and cells. F to H, pBIN-mGFP5-ER-transfected protoplasts and cells. D through H show ER network. I and J, pRTL2-GFP-transfected protoplasts and cells. GFP is cytosolic. Bars represent 10 _μ_m.

Figure 6.

Figure 6.

Electron micrographs taken of freeze-substituted transgenic and nontransgenic N. tabacum leaf segments following various treatments. Cell wall (cw) mitochondria (m), Golgi stacks (g), ER (er), and ribosomes (r) are indicated. The long black and arrows point to GFP-labeled ultrastructures, and the black and arrowheads point to BiP-labeled ultrastructures. A to D, mGFP5-ER transgenic cells. A and D, mGFP5-ER transgenic tobacco cells probed with both GFP (10 nm gold) and BiP (20 nm gold) antiserum. B, mGFP5-ER transgenic cells treated with buffer and secondary antisera have no label in the ER. C, mGFP5-ER transgenic cells treated with GFP antisera show label associated with the ER. E, GFP:TGBp2 transgenic cell treated with buffer and secondary antisera. F, GFP:TGBp3 transgenic cell treated with buffer and secondary antisera. G, Nontransgenic cell treated with BiP antiserum. Bars represent 0.5 _μ_m.

Figure 7.

Figure 7.

Electron micrographs of freeze-substituted GFP:TGBp2 and GFP:TGBp3 transgenic N. tabacum leaf segments with various treatments. The 10-nm gold particles associated with GFP antiserum are indicated by long black arrows. The 20-nm gold particles associated with BiP antiserum are indicated by black arrowheads. The mitochondria (m), chloroplast (chl), cell wall (cw), Golgi apparatus (g), ER (er), and ribosomes (r) are indicated. A to C, GFP:TGBp2 transgenic cells show vesicles. Some vesicles lay along the cell wall. D to F, GFP:TGBp3 transgenic cells. A, C, D, and E, Samples treated with GFP antisera. B and F, Samples treated with GFP and BiP antiserum. Bars represent 0.5 _μ_m.

Figure 8.

Figure 8.

Confocal images are of N. benthamiana leaves bombarded with pRTL2-mGFP5-ER, GFP:TGBp2, and GFP:TGBp3 and treated with BFA or latrunculin B. Untreated cells transformed with mGFP5-ER (A); GFP:TGBp3 (B); or GFP:TGBp2 (C). D to F, mGFP5-ER-, GFP:TGBp3-, and GFP:TGBp2-expressing leaves treated with BFA, respectively. G to I, mGFP5-ER-, GFP:TGBp3-, and GFP:TGBp2-expressing leaves treated with latrunculin B, respectively. Arrowheads point to vesicles. Bars represent 10 _μ_m.

Figure 9.

Figure 9.

Confocal images showing localization of GFP:TGBp2 with actin, Golgi, and localization of GFP:TGBp1. A to C, Cells expressing GFP:TGBp2 and DsRed:Talin. Arrows point to vesicles along actin filaments. D to F, Cells expressing GFP:TGBp2 and GFP:Talin. Yellow arrowheads point to vesicles. F, Vesicles in cells treated with latrunculin B. G to J, Tobacco epidermal cells cobombarded with GFP-TGBp2 and DsRed-ST. Note that the GFP-TGBp2 vesicles (arrowhead) do not colocalize with the DsRed-ST decorated Golgi (arrows). K, Cells expressing GFP:TGBp2. L, Cells expressing GFP:TGBp1. Bars represent 10 _μ_m.

Figure 10.

Figure 10.

Model linking the ER stress response with virus movement. TGBp2 (black horseshoe) and TGBp3 (black bar) are located in the ER. TGBp2 is also located in ER-derived vesicles located at the periphery of the cell. During PVX infection, TGBp2 is located in vesicles and in the ER in cells located at the infection front and at the center of the infection foci (this study). We do not know whether TGBp3 colocalizes with TGBp2 in the ER-derived vesicles during PVX infection. The ER traverses the plasmodesmata and is a rich store of Ca2+ ions. Calmodulin (indicated by gray sphere with a bent arm) is resident in the ER and plasmodesmata and is one example of a factor that controls fluctuations of Ca2+ across the ER. The ER-derived vesicles line the cell wall and may also be Ca2+ stores. Unconventional myosin VIII (gray bars) is a component of plasmodesmata that regulates gating. Mobilization of calcium, perhaps as a result of ER stress, controls unconventional myosin VIII activities within the plasmodesmata. ER stress may be caused by TGBp2-induced ER reorganization. We do not yet know whether other PVX proteins associate with the TGBp2-induced vesicles, although it is known that TGBp2 and TGBp3 from PMTV colocalize in vesicles (Haupt et al., 2005). If the viral movement complex is associated with the TGBp2-induced vesicles, then it is possible the vesicles move across the plasmodesmata similar to the TMV VRCs. It is also possible that the viral movement complex is exported from the ER (or vesicles) and then moves across the plasmodesmata. Further research is needed to determine the role of the ER, ER stress, and calcium signaling in virus cell-to-cell transport. Arrow at the bottom indicates direction of virus movement from one cell into the adjacent cell.

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