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Planta (1999) 208: 392±400 Transport of virally expressed green ¯uorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A Petra Boevink1, Barry Martin2, Karl Oparka1, Simon Santa Cruz1, Chris Hawes2 1 Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK School of Biological and Molecular Sciences, Oxford Brooke University, Gipsy Lane, Oxford, OX3 0BP, UK 2 Received: 8 October 1998 / Accepted: 16 November 1998 Abstract. Potato virus X (PVX) has been used as an expression vector to target the green ¯uorescent protein (GFP) from the jelly®sh Aequorea victoria to the endoplasmic reticulum (ER) of tobacco (Nicotiana clevelandii L.) leaves. Expression of free GFP resulted in strong cytoplasmic ¯uorescence with organelles being imaged in negative contrast. Translocation of GFP into the lumen of the ER was mediated by the use of the sporamin signal peptide. Retention of GFP in the ER was facilitated by the splicing of the ER retrieval/ retention tetrapeptide, KDEL to the carboxy terminus of GFP. Fluorescence of GFP was restricted to a labile cortical network of ER tubules with occasional small lamellae and to streaming trans-vacuolar strands. Secretion of ER-targeted GFP was inhibited both by cold shock and low concentrations of the secretory inhibitor brefeldin A. However, both prolonged cold and prolonged incubation in brefeldin A resulted in the recovery of secretory capability. In leaves infected with the GFPKDEL construct, high concentrations of brefeldin A induced the tubular network of cortical ER to transform into large lamellae or sheets which reverted to the tubular network on removal of the drug. Key words: Endoplasmic reticulum ± Golgi apparatus ± ± Brefeldin A ± Green ¯uorescent protein ± Nicotiana ± protein secretion Introduction A number of dierent approaches have been taken to study the transport of secretory products through the Abbreviations: BFA = brefeldin A; GFP = green ¯uorescent protein; pTXS.P3C2 = potato virus X expression vector; PVX = potato virus X Correspondence to: C. Hawes; E-mail: [email protected]; Fax: 44 (1865) 483955 endomembrane system of plant cells. These started with the pioneering work of Pickett-Heaps (1968) who used radiolabelled probes in combination with electronmicroscope autoradiography to establish the role of the Golgi apparatus in cell wall matrix biosynthesis. More recently, anity labelling of secretory products in the endoplasmic reticulum (ER), Golgi, storage vacuoles and at the cell surface (Greenwood and Chrispeels 1985; Sherrier and VandenBosch 1994), and biochemical fractionation of the various components of the pathway (Brummell et al. 1990) have been the techniques of choice. However, it has been the use of heterologous expression systems, which permit the observation of foreign and chimeric proteins in model plant species, that has revolutionised our understanding of tracking and targeting of proteins within the secretory system (for reviews, see Chrispeels 1991; Neuhaus 1996). Thus, it has been demonstrated that plants use the H/KDEL Cterminal signal peptide for retention of proteins in the lumen of the ER (Denecke et al. 1992; Napier et al. 1992), and a range of peptide sequences for targeting proteins to the vacuole (Neuhaus 1996). Transport of secretory material post-Golgi is by means of vesicle vectors (Staehelin and Moore 1995; Hawes et al. 1996) and it is assumed that transport from the ER to the cis-Golgi is also by vesicles. However, evidence for the existence of ER-derived transport vesicles in higher plants is scant (Hawes and SatiatJeunemaitre 1996) with little in-vivo structural or biochemical data being available. In mammalian cells, ER-to-Golgi transport has been demonstrated by the use of the secretory inhibitor brefeldin A (BFA) which blocks vesicle transport between the ER and Golgi (Klausner et al. 1992). In plants, BFA has been shown to block transport from the ER and appears to inhibit secretion from the trans-Golgi (Matsuoka et al. 1995; Satiat-Jeunemaitre et al. 1996). To investigate the ERto-Golgi step of the secretory pathway in further detail a cell system synthesising and secreting a high level of foreign protein should prove invaluable. The green ¯uorescent protein (GFP) from the jelly®sh Aequorea victoria is a powerful tool for the study of gene P. Boevink et al.: Transport of virally expressed green ¯uorescent protein expression and for protein location using fusions of GFP to proteins of interest. The intrinsic ¯uorescence of the GFP allows non-destructive, in-vivo imaging of events within cells. Fused to the C-terminus of a neuroendocrine secretory protein, GFP has been used to image the secretory pathway (Kaether and Gerdes 1995) and the mobility of Golgi transferases in live, transfected HeLa cells (Cole et al. 1996). In plant cells, expression of a signal peptide-GFP-HDEL construct resulted in ¯uorescence of the ER in Arabidopsis thaliana roots (Haselo and Amos 1995). It has recently been shown that very high levels of foreign-protein expression can be obtained in plants using virus-based episomal vectors. A potato virus X (PVX) vector has been used to express both native GFP and chimeric GFP-protein fusions in tobacco leaves (Baulcombe et al. 1995; Oparka et al. 1996a). In combination with confocal microscopy this technique is proving invaluable for studying the targeting of proteins in vivo (Boevink et al. 1996, 1998; Oparka et al. 1996b). In a preliminary publication we demonstrated the targeting and retention of a GFP-KDEL construct to the ER of tobacco leaf cells using the patatin signal peptide (Boevink et al. 1996). Here we report on the increased eciency of the sporamin signal peptide for insertion of GFP-KDEL into the ER and on the secretion of GFP, without KDEL, to the apoplast. In addition, we show that both the inhibitor BFA and cold shock can inhibit transport of GFP downstream from the ER with a resulting build up of ¯uorescence in the ER lumen. Materials and methods Constructs. Standard DNA manipulation techniques were used (Sambrook et al. 1989). Overlap extension polymerase chain reaction (PCR; Higuchi et al. 1988) using mutagenic oligonucleotides was used to fuse the signal-peptide-encoding sequence (sp) from the sweet potato (Ipomoea batatas Lam.) storage protein, sporamin (Hattori et al. 1985), to the 5¢ end of the gfp gene (wild type) to give sp-GFP. ClaI and NsiI restriction-enzyme recognition sequences engineered into the mutagenic oligonucleotides allowed the overlap extension PCR product to be cloned into ClaI/NsiIdigested potato virus ´ expression vector (pTXS.P3C2; data not shown), giving rise to the plasmid pTXS.sp-GFP. A second construct, pTXS.sp-GFP-K, was made that also contained the coding sequence for the carboxy-terminal 11 amino acids from a heat-shock protein (HSP90) homologue, which include KDEL (Schroder et al. 1993), fused to the 3¢ end of the gfp gene (Fig. 1). In-vitro transcription and plant inoculation. Templates for transcription were prepared by digesting pTXS-GFP (Baulcombe et al. 1995), pTXS.sp-GFP and pTXS.sp-GFP-K with the restriction enzyme SpeI. In-vitro, run-o transcripts were synthesised from the templates using a T7 transcription kit (Ambion, Austin, Tex., USA). Leaves of approximately 4-week-old Nicotiana clevelandii L. plants were inoculated with transcripts by rubbing the transcription product derived from 0.2 mg of template (per leaf) onto aluminium-oxide-dusted leaves. Note that progeny viruses on plants inoculated with in-vitro transcripts are referred to by substituting the pre®x PVX for the plasmid name pTXS. Immunochemical detection of GFP. Total protein preparations were made from infected leaves at 10±12 days post inoculate and from 393 Fig. 1. Insertion of modi®ed GFPs into the PVX vector to create pTXS.sp-GFP and PTX.sp-GFP-K. The PVX vector, pTXS.P3C2, and modi®ed GFPs are not drawn to scale. The viral cDNA is ¯anked by T7 RNA promoter and speI recognition sequences. Open boxes represent coding sequences and lines represent untranslated sequence. The genes in the PVX vector are, from left to right: the replicase (REP), the triple-gene-block proteins, represented by their approximate molecular weights in kDa (25, 12, 8), and the coat protein (CP). pTXS.P3C2 contains a duplication of the coat protein subgenomic promoter (black triangles) and a multiple cloning site (magni®ed). The two modi®ed GFP genes with the sporamin signal peptide at the 5¢ terminus (SP) and with or without sequence encoding the KDEL peptide (K) at the 3¢ terminus were inserted independently into the vector between the ClaI and NsiI sites to give pTXS.spGFP-K and pTXS.sp-GFP, respectively healthy (non-inoculated) leaves by grinding them in one and a half times (w/v) grinding buer [100 mM Tris-HCl (pH 7.5), 10 mM KCl, 5 mM MgCl2, 0.4 M sucrose, 10% glycerol, 15 lM bmercaptoethanol]. The resulting mixture was centrifuged in a microfuge and the supernatant collected. Aliquots of the extract were taken and mixed with equal volumes of loading buer (1% glycerol, 1% SDS, 12.5 bg/ml bromophenol blue, 6.25 mM TrisHCl, 5 mM b-mercaptoethanol, pH 6.8). Intercellular ¯uid extracts were obtained from infected and healthy leaves by vacuumin®ltrating the leaves with water and centrifuging them at 4000 g for 2±3 min. Proteins in the extract were concentrated by mixing with Strataclean resin (Stratagene) and centrifuging. Loading buer was added directly to the precipitated resin. Samples with loading buer added were boiled for 5±10 min and loaded onto an SDS-PAGE gel. Gels were electrophoresed at 100 V for 4±5 h and the separated proteins were transferred to nitrocellulose membranes. Membranes were probed with anti-GFP (Santa-Cruz et al. 1996) and anti-PVX-coat-protein antibodies (Oparka et al. 1996a). Brefeldin A and cold treatments. Brefeldin A (Sigma) was kept as a 10 mg/ml stock solution in methanol. Explants about 3 mm square were taken from infected areas of leaves from plants inoculated with pTXS.sp-GFP and pTXS.sp-GFP-K transcripts and ¯oated on 10 or 100 lg/ml BFA, or 1% methanol in distilled water, for 4± 12 h. Eects of BFA on the distribution of GFP were monitored by confocal microscopy. The ecacy of the used BFA was tested on fresh leaf segments. For cold treatment, plants were held under continuous light at 4 °C for varying lengths of time and segments of leaves excised from infected areas for confocal microscopy. 394 P. Boevink et al.: Transport of virally expressed green ¯uorescent protein Fig. 2. Comparison of the ¯uorescence of lesions produced by PVX.GFP, PVX.spGFP-K and PVX.sp-GFP. Nicotiana clevlandii leaves infected with PVX.GFP (left), PVX.sp-GFP-K (centre) or PVX.sp-GFP (right) at 6 d post infection. The PVX.spGFP lesions are noticeably less bright than the control PVX-GFP. The PVX.sp-GFP lesions are visible as darker regions on the leaf. Some auto¯uorescence due to damage is also visible on this leaf. Bar = 1 cm Confocal microscopy. Leaf pieces were viewed, usually at approx. 6±8 days post inoculation, with a Bio-Rad MRC 1000 laser scanning microscope and all images are of epidermal cells of the adaxial leaf surface. The methods were as described by Baulcombe et al. (1995). Electron microscopy. Segments of leaf 2 mm square were excised under ®xative containing 2% glutaraldehyde and 1% paraformaldehyde in 0.1 M sodium cacodylate buer (pH 6.9), transferred to fresh ®xative for 1 h under vacuum and post-®xed for 2 h in 1% aqueous osmium tetroxide. After dehydration in a graded water/ ethanol series, material was embedded in Spurr resin (Spurr 1969), sectioned with an MT7000 ultramicrotome (RMC, Tucson, Arizona), stained with uranyl acetate and lead citrate and observed with a 1200EXII (JEOL, Tokyo, Japan) transmission electron microscope. Results Expression of GFP constructs in tobacco leaves. To target GFP into the secretory pathway the coding sequence for the 21-amino-acid signal peptide of the sweet-potato storage protein sporamin (Hattori et al. 1985) was fused to the amino-terminus of the gfp gene. A second construct was made, to retain the GFP in the ER, with the sporamin signal peptide fused to the amino-terminus and a KDEL retrieval/retention signal fused to the carboxy-terminus of the gfp gene (Boevink et al. 1996). The recombinant gfp genes were cloned into the PVX expression vector pTXS.P3C2 to give pTXS.sp-GFP and pTXS.sp-GFP-K respectively (Fig. 1). Circular, green ¯uorescent lesions containing cells expressing GFP were visible under a 395-nm UV lamp on Nicotiana clevelandii leaves inoculated with pTXS.sp-GFP-K after 4 d. The pTXS.sp-GFP-K lesions showed a lower level of ¯uorescence than PVX.GFP control leaves (Fig. 2), whilst lesions of PVX.sp-GFP leaves were only visible as slightly darker regions on the leaf (Fig. 2). The viral lesions increased in size with time and systemic symptoms, and also systemic ¯uorescence in the case of PVX.sp-GFP-K, were observed at about 7 d post- inoculation, indicating that the modi®ed viruses were not impaired in cell-to-cell or long-distance movement. Leaf epidermal cells infected with PVX.sp-GFP and viewed by confocal microscopy, showed very faint GFP ¯uorescence that appeared to be ER (Fig. 3C). This can be compared with the very high levels of cytoplasmic GFP ¯uorescence in PVX-GFP cells with no ER signal peptide, where the same microscope contrast and brightness settings were used to record the image (Fig. 3A). In these PVX-GFP cells cytoplasmic organelles could be seen in negative contrast (Fig. 3B). Occasionally, in some older, heavily infected areas of leaves, apoplastic GFP ¯uorescence was also detected, indicating secretion of the sp-GFP (Fig. 3D). Cells infected with PVX.sp-GFP-K, had the same subcellular distribution of GFP as that previously described for the construct, PVX.p-GFP-K containing the patatin signal peptide (Boevink et al. 1996). Fluorescence was restricted to the ER, which had two predominant distributions: (i) in a labile cortical network of tubules with occasional small lamellae (Fig. 3E,F) and (ii) in bright transvacuolar strands (not shown). Within both these systems small motile ¯uorescent inclusions were observed. The cortical network of ER is restricted to a thin layer of cortical cytoplasm approximately 0.1±0.3 lm thick (Fig. 4). Visually, the level of ¯uorescence in the ER of PVX.sp-GFP-K-infected cells was substantially higher than that previously reported with PVX.p-GFP-K (Boevink et al. 1996) although with the viral expression system this could not be quanti®ed. This result was con®rmed by the observation that PVX.sp-GFP-K lesions were visually ¯uorescent under a hand-held U/ V lamp, whereas PVX.p-GFP-K lesions were not (data not shown). The total amount of GFP ¯uorescence observed by confocal microscopy associated with PVX.sp-GFP was much lower than in PVX.sp-GFPK- or PVX.GFP-infected cells. This was in agreement with the dierences in ¯uorescence seen at the macroscopic level with UV illumination (Fig. 2). P. Boevink et al.: Transport of virally expressed green ¯uorescent protein Fig. 3A±F. Expression of GFP in tobacco leaf epidermal cells. A Epidermal cell in a leaf infected with PVX-GFP demonstrating high a level of expression of cytoplasmic GFP (compare with C). B Detail of a PVX-GFP epidermal cell showing that organelles (arrows) can be observed in negative contrast. C Epidermal cell in a leaf infected with PVX.sp-GFP. This construct results in extremely low levels of ¯uorescence in the ER which can only be observed with high laser power and high gain settings on the confocal microscope. Note that for comparative purposes the contrast and brightness settings for A and C were identical. D A heavily infected PVX.sp-GFP leaf epidermis showing apoplastic accumulation of GFP. E Low magni®cation of a leaf infected with PVX.sp-GFP-K showing GFP ¯uorescence of the cortical network of ER in several epidermal cells. Uninfected guard cells with auto¯uorescent central cell walls and chloroplasts can be seen in the centre of the image. F Epidermal cells of a leaf infected with PVX.sp-GFP-K showing detail of GFP ¯uorescence in the cortical network of ER. Bars = 15 lm (A±C, F), 30 lm (E), 50 lm (D) 395 Western Blotting. Further con®rmation that the GFP expressed from PVX.sp-GFP was secreted into the extracellular space was obtained from western blotting of intercellular ¯uid extracts from infected leaves (Fig. 5). Intercellular ¯uid extracts were prepared by vacuum-in®ltrating leaves with water followed by lowspeed centrifugation to extract the in®ltrated water. Proteins in the extracts were concentrated using a protein-binding resin. Relative to the total amount of GFP in PVX.sp-GFP-infected leaves (a very faint GFP band was visible in the total protein extract on the original membrane), the majority of GFP appeared to be extracellular (Fig. 5b, lanes 3 and 7). Direct quantitative comparisons between the dierent constructs were not possible because of variation in the levels of viral infection from plant-to-plant and leaf-to-leaf. Blots of viral coat protein from the dierent infections indicated that PVX-GFP plants contained the highest levels of 396 P. Boevink et al.: Transport of virally expressed green ¯uorescent protein Fig. 4. Transmission electron micrograph of a tobacco leaf epidermal cell demonstrating the thin layer of cortical cytoplasm. 120 kV, bar = 4 lm Insert: high magni®cation showing cortical endoplasmic reticulum (ER) in epidermal cell cytoplasm. ´45 000 coat protein, and therefore virus, followed by PVX.spGFP-K plants with PVX.sp-GFP plants showing the lowest levels (Fig. 5a, lanes 5±3 and 9±7). There also appeared to be substantially less GFP in PVX.sp-GFPand PVX.sp-GFP-K-infected tissue relative to the amount of virus present and compared to the PVX.GFP control. It was thought that a proportion of the GFP in the endomembrane system or the extracellular matrix may have been precipitated with the cell debris in total protein extractions, thus preventing true quantitative comparisons between the dierent viral constructs. Cold-induced inhibition of secretion. To determine whether cold shock could inhibit secretion in tobacco cells, PVX.sp-GFP-infected plants showing systemic symptoms were placed at 4 °C. The GFP ¯uorescence was visible in the ER after 8±12 h (Fig. 6A compare with Fig. 3C). The ER-localised ¯uorescence gradually diminished when plants were returned to room temperature until, after 4±6 h, the cells had similar ¯uorescence to the untreated PVX.sp-GFP-infected cells (Fig. 6B compare with Fig. 3C). The ER-localised ¯uorescence also diminished in plants left in the cold for more than 12±18 h (data not shown). Incubation at 10 °C had no eect on PVX.sp-GFP leaves (data not shown). Eects of BFA on the ER and on secretion. To determine whether the secretory inhibitor BFA would also induce a build up of GFP ¯uorescence in the ER, sp-GFPexpressing leaf segments were treated with 10 lg/ml of the drug for 8±12 h. These leaf segments showed an accumulation of GFP ¯uorescence in the ER (Fig. 7A), and on removal of the drug the ER-localised ¯uorescence gradually faded over 8 h (Fig. 7B). However, prolonged incubation of leaf segments in BFA (>18 h) also resulted in a gradual loss of the accumulated ¯uorescence in the ER, indicating a recovery of secretory ability in the presence of the drug (Fig. 7C). A similar treatment of PVX.sp-GFP-K-expressing leaves had little eect on the morphology of the ER. However, in leaf segments treated for 1±4 h with 100 lg/ml BFA, dramatic but reversible morphological changes to the ER were observed. The cortical ER gradually changed from the network of tubules typically observed, to large sheets or lamellae punctured by irregularly sized holes (Fig. 7D,E). On removal of the BFA, the tubular ER network, as seen in untreated cells, re-formed in 1±2 h (Fig. 7F). Discussion The signal peptide plus a KDEL sequence was required for the insertion and retention of GFP in the ER, although it is likely that some protein escaped the retention mechanism and was secreted. Green ¯uorescent protein that was targeted to the ER by the fused signal peptide, but which did not contain KDEL was barely visible in the ER and appeared to be largely secreted. We would not expect any retention mechanism for GFP other than the added KDEL peptide, as it is a protein foreign to the plant and when expressed without a signal sequence is normally found in the cytoplasm and nucleus (Baulcombe et al. 1995). The PVX signal peptide-GFP-KDEL construct made with the sporamin signal peptide was markedly brighter than the construct previously made that used the patatin signal peptide (Boevink et al. 1996). This suggests that the two signal peptides may dier in the eciency with which they direct protein translocation into the ER. In PVX.sp-GFP-infected cells the lack of overall ¯uorescence may indicate that, in the majority of the GFP molecules, the ¯uorophore had not had time to form in order for the protein to become ¯uorescent before it was P. Boevink et al.: Transport of virally expressed green ¯uorescent protein Fig. 5a,b. Western blots of total protein and intercellular ¯uid extracts from tobacco leaves. a Western blot probed with anti-PVX coat protein antibody. Lanes 1±9 contain: the PVX coat protein standard (1), total protein extract from a healthy plant (2), plants infected with PVX.sp-GFP (3), PVX.sp-GFP-K (4), and PVX.GFP (5), and intercellular ¯uid extracts from a healthy plant (6), and plants infected with PVX.sp-GFP (7), PVX.sp-GFP-K (8), and PVX.GFP (9). Equal amounts of extract were loaded per lane, except for lane 5 where half as much was loaded. b Western blot of the same extracts shown in a but probed with anti-GFP antibody. Lanes 1±9 contain: GFP standard (1), total protein extracts from a healthy plant (2), and plants infected with PVX.sp-GFP (3), PVX.sp-GFP-K (4), and PVXGFP (5), plus intercellular ¯uid extracts from a healthy plant (6), and plants infected with PVX.sp-GFP (7), PVX.sp-GFP-K (8), and PVX.GFP (9). Twice as much extract, compared with blot a, was loaded per lane. Note that in plants infected with PVX.sp-GFP, GFP can only be detected in intercellular ¯uid extracts secreted. It is known that for native GFP to fold and mature into the ¯uorescent form the chromophore has to cyclise, be oxidised and be encapsulated within the folded protein, a process which can take from 2±4 h for wild-type GFP (Cubitt et al. 1995). However, it is possible that the concentration of GFP in the ER lumen was extremely low because it was continually being secreted. The very low levels of ¯uorescence in PVX.sp-GFP-infected leaves may indicate that most GFP molecules which are secreted into the apoplast before the ¯uorophore has cyclised and the protein folded, are unable to mature in the apoplastic environment. In some older leaves with substantial viral infection, when high levels of GFP production would be expected, extracellular ¯uorescence of GFP could be detected (Fig. 3D). This, combined with the fact that GFP has an inherent stability (Cubitt et al. 1995), makes the alternative explanation that the protein is proteolytically cleaved in the cell wall and/or apoplastic space less likely. 397 Fig. 6A,B. Eect of cold on GFP secretion in tobacco leaves. A After 12 h at 4 °C, GFP ¯uorescence in the ER of PVX.sp-GFP infected leaves is apparent, indicating inhibition of export of GFP from the ER. B After returning cold-shocked plants to room temperature for 12 h most ¯uorescence is lost from the ER, indicating a resumption of secretion. Note the bright GFP-containing viral inclusion in the cell. Bars = 20 lm (A), 10 lm (B) Cold shock. A short period of cold shock appeared to block transport from the ER to the Golgi, allowing GFP ¯uorescence to build up to easily detectable levels in the ER of PVX.sp-GFP-infected cells in a manner similar to BFA treatment of the same leaves. Thus, it appears that one response of tobacco plants to cold is an inhibition of post-ER secretion. However, when left at low temperatures for longer periods the plants acclimatised to the new conditions and resumed secretion. Whilst temperature blocks to secretion are widely reported in mammalian cell systems (Roman and Garo 1985; Presley et al. 1997), there is little information of the eect of temperature on the higher-plant secretory pathway. Some reports have suggested that a block of vesicle and lipid transfer between the ER and Golgi may take place at a temperature as high as 12 °C (SturboisBalcerzak et al. 1995), although when PVX.sp-GFP tobacco plants were incubated at 10 °C we detected no increase in ER ¯uorescence. Eects of BFA on secretion and ER morphology. Treatment of PVX.sp-GFP-infected cells with relatively low concentrations of BFA resulted in accumulation of GFP in the ER whereas in control leaves minimal ¯uorescence in the ER was detected. This is a clear 398 P. Boevink et al.: Transport of virally expressed green ¯uorescent protein Fig. 7A±F. Eects of BFA on secretion of GFP and ER morphology in tobacco leaves. A Fluorescence of GFP in the lumen of the ER of a PVX.sp-GFP-infected leaf after incubation in 10 lg/ml BFA for 12 h, indicating inhibition of secretion of GFP from the ER. Note several bright viral inclusions are also present in the cortical cytoplasm. B Leaf epidermal cell after removal of BFA and incubation in distilled water for 8 h. Secretory activity has resumed and ER ¯uorescence is lost. Compare with Fig. 3B. C Leaf epidermal cell after prolonged incubation (18 h) in 10 lg/ml BFA. Note that ER ¯uorescence is again lost, indicating recovery of secretory capability in the presence of the drug. Note the guard cells to the left of the micrograph. D Early stages of morphological changes to the cortical ER network of a PVX.sp-GFP-K-infected leaf after treatment for 2±3 h with 100 lg/ml BFA. Note the formation of lamellae of ER compared to untreated leaf cells (Fig. 3D). E An epidermal leaf cell after 4 h treatment with 100 lg/ml BFA. Note the conversion of the cortical ER network in two cells into fenestrated sheets of membrane. Small ¯uorescent bodies can be seen on the surface of the membrane. F Recovery of the cortical ER network 6 h after removal of the BFA. Bars = 15 lm in-vivo demonstration of the inhibition of secretion in plant cells by BFA. Thus, the lack of substantial ER ¯uorescence in PVX.sp-GFP-infected cells (Fig. 3C) must re¯ect the fact that GFP was indeed secreted. We assume that the block is in a transport step between the ER and the cis-Golgi, although from these experiments a trans-Golgi blockage of secretion cannot be ruled out. However, we did not observe a build up of ¯uorescent structures that could be attributed to GFP accumulation in the Golgi stacks, the bright spots of ¯uorescence on the ER being smaller than would be expected for Golgi bodies (Fig. 7E). This result is also supported by the fact that recently we have shown that a similar BFA treatment can result in the destruction of GFP-tagged tobacco leaf Golgi (Boevink et al. 1998). It has previously been shown that BFA treatment inhibits ER-toGolgi transport (Matsuoka et al. 1995) and secretion in plant cells (Driouich et al. 1993; Satiat-Jeunemaitre and P. Boevink et al.: Transport of virally expressed green ¯uorescent protein Hawes 1994; Satiat-Jeunemaitre et al. 1996) and also results in a trans-Golgi accumulation of secretory products (Satiat-Jeunemaitre and Hawes 1993). However, this is the ®rst in-vivo visual indication that blockage of the secretory pathway can lead to an accumulation of secretory product in the lumen of the ER. It is also interesting to note that prolonged incubation of leaf segments in BFA resulted in eventual loss of ¯uorescence of the ER. It is assumed that this is attributable to the secretory machinery being re-established in the presence of the drug. This time-dependent acquisition of resistance to BFA has also been reported in maize roots where Golgi stacks vesiculated in the presence of a high concentration of BFA (2 h in 100 lg/ ml BFA) but with time eventually re-formed (Steele 1997). This work also demonstrated that BFA retained activity after material had been incubated in the drug for 24 h, supporting our conclusion that in tobacco leaves we are observing some form of induced resistance to prolonged exposure to BFA. The application of high concentrations of BFA induced rapid and dramatic changes to the morphology of the ER which were reversible on removal of the drug. The change from the cortical network of tubules to large ¯at membrane lamellae was dierent from changes seen in the ER of BFA-treated root cells (Satiat-Jeunemaitre et al. 1996). Here, it was shown that the ER in meristematic root tips cells, when visualised after immuno¯uorescence detection with an HDEL antibody, aggregated into tubular and lamellar clusters in the cytosol. It has previously been demonstrated that the morphology of cortical ER is extremely sensitive to pharmacological (e.g. calcium chelators, cytoskeletal inhibitors) and environmental perturbations (e.g. heat stress), showing considerable pleomorphy between tubular and lamellar forms (Quader et al. 1996). However, such a dramatic morphological change in response to a secretory inhibitor has not previously been reported. One explanation is that in the viral expression system, high levels of GFP-KDEL accumulate in the lumen of the ER, saturating the retention/retrieval mechanism of the cell. Treatment with BFA would then block this secretion of excess protein and lead to a further build up in the ER, resulting in a dramatic change in morphology and lumen volume to accommodate the GFP-KDEL and any other secretory proteins. On removal of the drug, secretion resumes and the morphology of the ER reverts to the normal state. Alternatively, a direct action of the drug on the ER membrane cannot be excluded as it has been reported that BFA can in¯uence the synthesis and transport of lipids from the ER (Slomiany et al. 1993). Associated with the sheets of ER in BFA-treated cells were small irregular ¯uorescent structures. It is assumed that these are either aggregations of GFP or virus protein associated with GFP. It is unlikely that they represent Golgi stacks, or the remains of Golgi stacks, as in the tobacco leaf system the Golgi is extremely sensitive to shorter (less than 30 min) treatments of BFA (Boevink et al. 1998). 399 We thank Mr. Denton Prior (SCRI) for help with confocal microscopy, Dr. L. Faye (CNRS, Rouen, France) for supplying sporamin constructs and Dr. B. Satiat-Jeunemaitre (CNRS, Gifsur-Yvette, France) for critical reading of the manuscript. This work was supported by a grant from the Leverhulme Trust. 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