Silencing of Nicotiana benthamiana Neuroblastoma-Amplified Genecauses ER stress and cell death
- Jae-Yong Lee†1,
- Sujon Sarowar†1,
- Hee Seung Kim1,
- Hyeran Kim2,
- Inhwan Hwang2,
- Young Jin Kim3 and
- Hyun-Sook Pai1Email author
© Lee et al.; licensee BioMed Central Ltd. 2013
Received: 8 January 2013
Accepted: 23 April 2013
Published: 27 April 2013
Neuroblastoma Amplified Gene (NAG) was identified as a gene co-amplified with the N-myc gene, whose genomic amplification correlates with poor prognosis of neuroblastoma. Later it was found that NAG is localized in endoplasmic reticulum (ER) and is a component of the syntaxin 18 complex that is involved in Golgi-to-ER retrograde transport in human cells. Homologous sequences of NAG are found in plant databases, but its function in plant cells remains unknown.
Nicotiana benthamania Neuroblastoma-Amplified Gene (NbNAG) encodes a protein of 2,409 amino acids that contains the secretory pathway Sec39 domain and is mainly localized in the ER. Silencing of NbNAG by virus-induced gene silencing resulted in growth arrest and acute plant death with morphological markers of programmed cell death (PCD), which include chromatin fragmentation and modification of mitochondrial membrane potential. NbNAG deficiency caused induction of ER stress genes, disruption of the ER network, and relocation of bZIP28 transcription factor from the ER membrane to the nucleus, similar to the phenotypes of tunicamycin-induced ER stress in a plant cell. NbNAG silencing caused defects in intracellular transport of diverse cargo proteins, suggesting that a blocked secretion pathway by NbNAG deficiency causes ER stress and programmed cell death.
These results suggest that NAG, a conserved protein from yeast to mammals, plays an essential role in plant growth and development by modulating protein transport pathway, ER stress response and PCD.
KeywordsbZIP28 ER stress gene expression Promoter-GUS fusion Protein transport assay Virus-induced gene silencing
Programmed cell death (PCD) is a genetically defined process associated with distinctive morphological and biochemical characteristics, and is an integral part of the life cycle of multicellular organisms [1, 2]. In plants, PCD occurs during developmental processes and in response to abiotic and biotic stresses [3, 4]. The major PCD signaling pathways involve mitochondria and plasma membrane receptors, although it has recently been shown that ER stress caused by impaired ER function can also induce apoptotic pathways in animals and plants [5–7].
The ER performs several important functions, including protein targeting and secretion, vesicle trafficking, and membrane biogenesis, and its proper function is essential to cell survival. Perturbations in ER homeostasis disrupt folding of proteins, leading to accumulation of unfolded proteins and protein aggregates. This condition is called ER stress and is detrimental to cell survival [5, 8]. Under conditions of ER stress, a cell activates a signal transduction pathway termed the unfolded protein response (UPR) to limit the damage and maintain ER homeostasis [7, 9, 10]. Prolonged and excessive ER stress induces apoptosis, evidenced by DNA fragmentation, cytochrome c release, and induction of caspase activity . In mammals, the ER stress-induced apoptotic pathway involves cross-talk between ER and mitochondria through Bcl-2 family members Bcl-2, Bax and Bak, which are localized in both mitochondria and ER . Recent reports suggest that in animal cells, the mitochondrial apoptotic pathway mediated by Apaf-1 is an integral part of ER stress-induced apoptosis [2, 5, 11]. In plants, treatment with tunicamycin, an inhibitor of N-linked protein glycosylation, and cyclopiazonic acid, a blocker of plant ER-type IIA calcium pumps, elicits ER stress, followed by activation of PCD with typical apoptotic morphology [6, 12]. However, the mechanisms of UPR and ER stress-induced PCD in plants remain largely unknown. One mechanism of ER stress response in Arabidopsis involves membrane-associated bZIP transcription factors; following ER stress, bZIP28 and bZIP60 are processed and their released N-terminal domains are translocated to the nucleus to upregulate the expression of ER stress response genes including BiPs (ER chaperones), BI-1 (Bax-Inhibitor 1), PDI (protein disulfide isomerase), and calnexin [13–15].
Genomic amplification (3 to 300 copies) of N-myc oncogene in human neuroblastoma correlates with aggressive tumor growth and poor prognosis . Neuroblastoma Amplified Gene (NAG) was first identified as a gene co-amplified with the N-myc gene, although NAG is widely expressed in normal human tissues and its homologous sequences are found in plant databases [16, 17]. Recently, it has been shown that NAG is localized in ER and is a component of the syntaxin 18 complex that is involved in Golgi-to-ER retrograde transport using human cell lines . In this study, we investigated in planta functions of NAG in Nicotiana benthamiana. We showed that N. benthamiana NAG (NbNAG) played a role in protein transport pathway, and NbNAG deficiency caused ER stress and cell death, suggesting its essential role in plant growth and survival.
Identification of NbNAG
Functional genomics using virus-induced gene silencing (VIGS) revealed that silencing of the N. benthamiana homolog of Neuroblastoma Amplified Gene (NAG), designated NbNAG, results in growth arrest and acute plant death. The ~7.4 kb full-length NbNAG cDNA encodes a polypeptide of 2,409 amino acids with a predicted molecular mass of 270,589.33 Da (Additional file 1: Figure S1). Database searches identified closely related genes in human, mouse, zebrafish, C. elegans, Arabidopsis and rice. The NAG homolog encodes a protein of ~2,400 amino acids in Arabidopsis (At5g24350), rice (NP_001066451), and human (NP_056993), and is a single-copy gene in all three genomes. Our analyses showed that there is no null mutant of NAG in Arabidopsis T-DNA insertion lines. The NbNAG protein contains the Sec39 domain (residues 595–1126) that has been implicated in ER-Golgi trafficking in yeast . Overall, NbNAG shows 49%, 42% and 29% sequence identity to the NAG homologs from Arabidopsis, rice, and human, respectively. NbNAG sequence was aligned with those of the NAG homologs from Arabidopsis and rice (Additional file 1: Figure S1).
ER localization of NbNAG in tobacco BY-2 cells
Expression of Arabidopsis NAG
VIGS phenotypes and suppression of endogenous NbNAGtranscripts
VIGS with TRV:NAG(N1) and TRV:NAG(N2) constructs resulted in growth arrest and acute plant death (Figure 3C-F). Necrotic lesions were evident in young leaves around the shoot apex at 15–16 days after infiltration (DAI) (Figure 3G). At 25 DAI, the shoot apex was completely abolished with no stem growth or new leaf formation (Figure 3D, E). The lesions progressively expanded, leading to premature death of the plants. Root growth was also affected by NbNAG VIGS at 15 and 25 DAI (Figure 3H, I). The petiole of the fourth leaf above the infiltrated leaf, and the stem where the fourth leaf above the infiltrated leaf was attached were cross-sectioned freehand and observed under light microscopy (Figure 3J-M). The localized brown pigment in the petiole (Figure 3K, cf. control in J) and the stem (Figure 3M, cf. control in L) at 20 DAI indicates cell death in the vasculature of TRV:NAG lines, particularly in the cambium. Fluorescence microscopy revealed that TRV:NAG lines accumulated large amounts of autofluorescent secondary metabolites, which were also observed at infection sites during hypersensitive cell death , in the stem vasculature (Figure 3O), while TRV control exhibited the fluorescence only in the xylem tracheary elements (Figure 3N).
Analysis of programmed cell death phenotypes
During apoptosis in animal cells, activation of the cell death pathway is initiated by modification of mitochondrial membrane permeability [3, 4]. Mitochondrial membrane potential of leaf protoplasts from VIGS lines was monitored by TMRM (Tetramethylrhodamine methyl ester) fluorescent probes that accumulate in mitochondria in proportion to the mitochondrial membrane potential . The average TMRM fluorescence of TRV:NAG protoplasts was ~4-fold lower than that of TRV control at 20 DAI, indicating reduced mitochondrial membrane potential (Figures 4D, E, Additional file 1: Figure S5A), but there was no difference in TMRM fluorescence between TRV and TRV:NAG lines at 10 DAI (Additional file 1: Figure S2B, C). Chlorophyll autofluorescence was not affected in TRV:NAG protoplasts at either 10 DAI (Additional file 1: Figure S2B, D) or 20 DAI (Figures 4D, F, Additional file 1: Figure S5A). To test whether reactive oxygen species (ROS) are involved in the cell death phenotype of TRV:NAG plants, leaf protoplasts prepared from VIGS lines (20 DAI) were incubated with H2DCFDA that becomes activated in the presence of H2O2 to produce green fluorescence. Accumulation of fluorescent H2DCFDA in TRV:NAG protoplasts was ~4.2-fold higher than in TRV control, indicating H2O2 accumulation (Figures 4G, H, Additional file 1: Figure S5A).
Ultrastructural analyses of the cell death phenotype
Light (Additional file 1: Figure S3A, B) and transmission electron microscopy (TEM) (Additional file 1: Figure S3C-L) of transverse leaf sections revealed degenerating spongy mesophyll cells at early and late stages in TRV:NAG lines, in contrast to TRV control cells at 25 DAI. TEM also showed disintegrating chloroplasts (Additional file 1: Figure S3E, F, J) and mitochondria (Additional file 1: Figure S3L), ruptured vacuoles (Additional file 1: Figure S3E, F), and abnormal nuclei (Additional file 1: Figure S3H) in TRV:NAG lines, compared with TRV control (Additional file 1: Figure S3C, G, I, K). These results suggest that the NbNAG-induced cell death involves vacuole collapse leading to enzymatic degradation of organelles and cell contents, as demonstrated in PCD induced by other stimuli .
NbNAG deficiency inhibits intracellular protein transport
We next examined the effects of NbNAG depletion on the secretory pathway using invertase:GFP, a chimeric protein consisting of full-length secretory invertase and GFP , as a reporter (Figure 5B). After transformation with the invertase:GFP construct, green fluorescent signals were not detectable in TRV protoplasts, indicating that invertase:GFP was secreted into the medium as previously observed . However, invertase:GFP signal was readily detected in TRV:NAG protoplasts (Figure 5B). To confirm this finding, proteins were extracted from the protoplasts and the incubation medium, and analyzed by western blot analysis with anti-GFP antibody (Figures 5F, Additional file 1: Figure S5B). In TRV controls, invertase:GFP (~120 kDa) was mainly detected in the medium, while a minor portion of the protein remained in the protoplasts. In contrast, in the TRV:NAG line the fusion protein was predominantly present in the protoplasts, suggesting that NbNAG depletion blocked the secretion of invertase:GFP into the medium.
Next, we examined ER-Golgi transport using an artificial ER marker protein, GKX . TRV protoplasts transformed with GKX exhibited a reticular network pattern of green fluorescence (Figure 5C) as observed previously . However, TRV:NAG protoplasts expressing GKX displayed a punctate and aggregated fluorescence signal, suggesting disruption of the ER network. Since GKX contains an N-glycosylation site, we examined the sensitivity of the N-glycans of GKX to endoglycosidase H (endo H) treatment. It has been reported that the N-glycans of ER proteins are sensitive to endo H, while the N-glycans of Golgi proteins are resistant due to modification [28, 29]. Protein extracts from TRV and TRV:NAG protoplasts were digested by endo H prior to western blotting with anti-GFP antibody (Figure 5G). In the TRV control, endo H digestion of GKX resulted in two bands, ~33 kDa endo H-sensitive GKX proteins (ER form) and ~36 kDa endo H-resistant proteins (Golgi form). These results indicate that a large part of GKX proteins was transported from ER to Golgi complex through anterograde trafficking. TRV:NAG lines also contained both the ER and the Golgi forms, but the ratio of the ER form to the Golgi form was higher than in TRV controls (Figures 5G, Additional file 1: Figure S5B). These results provide evidence that protein transport from ER to Golgi complex was inhibited by NbNAG deficiency.
NbNAG-silencing causes ER stress
To further observe ER morphology, we transiently expressed a chimeric lumenal protein, BiP:GFP, a fusion protein between BiP (lumenal binding protein) and GFP, in protoplasts from TRV and TRV:NAG lines (Figure 6B). At 10 DAI, the fluorescent signal of BiP:GFP was localized in a reticular membranous network throughout the cytoplasm of both TRV control and TRV:NAG lines. At 15 DAI, the signal was punctate and fragmented in TRV:NAG lines indicating a disrupted ER network, while TRV exhibited normal ER morphology (Figure 6B).
We next tested whether the chemical chaperone 4-phenyl butyric acid (PBA) alleviates NbNAG-induced cell death (Figure 6C-E). PBA relieves ER stress by directly reducing the load of misfolded proteins retained in the ER and has been shown to mitigate ER stress-induced cell death in mammals and plants [6, 30, 31]. Treatment with PBA (1 mM) partially rescued the growth retardation and cell death phenotypes of TRV:NAG lines (Figure 6C). Elevated ion leakage, an indicator of cellular membrane leakage, in TRV:NAG lines was also alleviated by PBA treatment in the 4th leaf above the infiltrated leaf (Figures 6D, Additional file 1: Figure S5A) and in the leaf near the shoot apex (Figures 6E, Additional file 1: Figure S5A). These results suggest that the defects in protein transport may cause ER stress in NbNAG-deficient cells.
Nuclear translocation of bZIP28
Neuroblastoma-Amplified Gene (NAG) encodes a large protein (~2,400 amino acids) containing the Sec39 domain in plants and mammals. In human neuroblastoma, NAG was found to be co-amplified with N-myc oncogene, of which genomic amplification correlates with aggressive tumor growth . Despite the marked size difference, the NAG ortholog of yeast appears to be Sec39p (Dsl3p), a cytosolic protein of ~82 kDa, that is peripherally associated with the ER membrane . Sec39p was first identified as an essential protein involved in ER-Golgi transport in a large-scale promoter shutoff analysis of essential yeast genes . Further characterization revealed that Sec39p is a component of the Dsl1p complex that also includes Dsl1p, Tip20p, and ER-localized Q-SNARE proteins [19, 33]. The Dsl1p complex is essential for retrograde traffic from the Golgi to ER, and consistently, Sec39 showed strong genetic interaction with other factors required for Golgi-ER retrograde transport [19, 34]. Interestingly, the temperature-sensitive sec39 mutants also exhibited defects in forward transport between the ER and Golgi , consistent with the results by Mnaimneh et al. . The phenotype can be explained by the fact that retrograde traffic plays an important role in recycling ER-resident proteins that have escaped from the ER [35, 36]. Thus, a blocked retrograde pathway can result in failure to retrieve these proteins and lead to a concomitant block in anterograde transport.
Mammals possess functional orthologs of the components of the yeast Dsl1p complex, despite a low degree of sequence conservation between the yeast and mammalian counterparts . Recently, it has been revealed that mammalian NAG is a subunit of the Syntaxin 18 complex involved in Golgi-to-ER retrograde trafficking and serves as a link between p31 and ZW10-RINT-1 in the mammalian ER fusion machinery . Interestingly, silencing of NAG in human cell lines using RNA interference did not substantially disrupt Golgi morphology or the ER-to-Golgi anterograde trafficking, but caused defects in protein glycosylation . In this study, we characterized in vivo effects of NAG deficiency in plants. NAG from N. benthamiana, Arabidopsis, and rice all encodes a ~2,400 kDa protein with the Sec39 domain that is mainly localized in ER, similar to the mammalian NAG. Knockdown of NbNAG using VIGS led to reduced protein transport between ER and Golgi, and a concomitant decrease in trafficking of the marker proteins to vacuole and to plasma membrane for secretion. Furthermore, NAG depletion in planta caused ER stress and PCD.
Our data suggest that ER stress caused by disrupted protein transport contributes to PCD activation in NbNAG-silenced plants. First, expression of the UPR-related genes such as BiP2, BiP5, BI1 (Bax inhibitor-1), HSP70, and CNK1 was up-regulated in NbNAG-silenced VIGS plants. BiPs encode ER-localized chaperones and have been used as marker genes for UPR activation in plants [12, 38], and similarly, expression of BI1, HSP70, and CNK1 was induced during tunicamycin-induced ER stress in Arabidopsis. Second, NbNAG deficiency caused proteolytic processing and nuclear relocation of bZIP28 transcription factor, which is normally associated with the ER membrane. It has been shown that bZIP28 processing is activated by ER stress-inducing agents such as tunicamycin and dithiothreitol, but not by salt stress . Third, the chemical chaperone PBA alleviated the growth retardation and cell death phenotypes of NbNAG VIGS plants. PBA suppresses ER stress and ER-mediated apoptosis by chemically enhancing the capacity of the ER to remove misfolded proteins in mammals and plants [6, 30, 31]. In addition, expression analyses of the Arabidopsis NAG promoter-GUS fusion gene showed that AtNAG promoter activity was stimulated in response to tunicamycin. Thus NbNAG appears to play a crucial role in protein transport during plant growth and development, and its deficiency causes ER stress response and subsequent activation of PCD. NbNAG-mediated cell death was first observed in young tissues containing actively dividing cells such as the shoot apex and vascular cambium before expanding to other tissues (Figure 3), consistent with the fact that young tissues have elevated demands for protein synthesis and transport.
Interestingly, mutations that inhibit the ER to Golgi trafficking have not always caused ER stress and PCD as observed in NbNAG VIGS plants. For example, a missense mutation in the COPII coat protein Sec24A caused the formation of aberrant tubular clusters of ER and Golgi membrane in Arabidopsis without inducing PCD . In yeast and mammals, the COPII coat forms transport vesicles on the ER surface for the ER-Golgi anterograde trafficking, and the COPII coat protein Sec24A is believed to have a specific role in cargo selection via site-specific recognition of cargo signals . In another case, overexpression of AtPRA1.B6, a prenylated Rab acceptor 1, resulted in inhibition of COPII vesicle-mediated anterograde trafficking but did not induce either ER stress or PCD . The lack of PCD activation in the Sec24A mutant may be linked to the fact that the missense mutation caused only a partial loss of Sec24A function, affecting the anterograde trafficking of only a subset of cargos . Total loss of Sec24A function instead led to an embryonic lethality, suggesting that the gene function is essential in Arabidopsis. Similarly, because AtPRA1.B6 functions as a negative regulator of the anterograde transport of only a subset of proteins at the ER, the effect of its overexpression appeared to be rather specific and limited . In this study, the severe consequences of NbNAG depletion suggest that NbNAG function in the protein transport pathway may be essential and/or common so that its deficiency severely disturbs the system. Alternatively, NbNAG may have additional, yet unidentified, functions, of which disruption induces PCD in a plant cell.
Although the underlying mechanism remains unclear, recent evidence indicates that mitochondria-dependent and –independent cell death pathways both play a role in ER stress-induced apoptosis [5, 8, 41]. Well-known regulators of mammalian apoptosis, such as the Bcl-2 family and caspases, are activated during ER stress [5, 41]. Bcl-2, Bax and Bak reside in the ER membrane as well as in the mitochondrial outer membrane, regulating homeostasis and apoptosis in the ER . Overexpression of Bcl-2 or deficiency of the proapoptotic proteins Bax and Bak confers protection against ER stress-induced apoptosis, indicating that Bcl-2 family members participate in the integration of apoptotic signals between the ER and mitochondria [42, 43]. In this study, NbNAG-induced cell death showed apoptotic hallmarks, such as nuclear DNA fragmentation, decreased mitochondrial membrane potential, and excessive production of reactive oxygen species. These apoptotic features are similar to the phenotypes of ER stress-induced PCD in Arabidopsis roots and soybean cell cultures caused by tunicamycin and cyclopiazonic acid, respectively [6, 44]. In particular, the decreased mitochondrial membrane potential strongly indicates involvement of mitochondria in NbNAG-induced PCD, at least at the stage we examined. It will be important to determine whether several pro-apoptotic pathways simultaneously function to commit the cell to death during ER stress, and how the different signals from the ER are integrated to activate PCD in plants.
Nicotiana benthamania Neuroblastoma-Amplified Gene (NbNAG) encodes an ER-localized protein of 2,409 amino acids that contains the secretory pathway Sec39 domain. NbNAG plays a role in protein transport pathway, and NbNAG deficiency resulted in ER stress and programmed cell death, presumably caused by a blocked secretion pathway. These results suggest that NAG, a conserved protein from yeast to mammals, plays an essential role in plant growth and development.
Virus-induced gene silencing
Virus-induced gene silencing was performed as described [22, 45, 46]. NbNAG cDNA fragments were PCR-amplified and cloned into the pTV00 vector containing part of the tobacco rattle virus (TRV) genome using the following NbNAG specific primers: NbNAG N1 (5'-aagcttatggaggaatcaact-3' and 5'-gggcccttggatcttgattga-3') and NbNAG N2 (5'-aagcttgttacagaatggaat-3' and 5'-gggcccagatatgccaagtcc-3'). The recombinant pTV00 plasmids and the pBINTRA6 vector containing RNA1 required for virus replication were separately transformed into Agrobacterium tumefaciens GV3101 strain. After grown to saturation, the Agrobacterium culture was centrifuged and resuspended in 10 mM MgCl2, 10 mM MES and 150 μM acetosyringone, and kept at room temperature for 2 h. Separate cultures containing pTV00 and pBINTRA6 were mixed in a 1:1 ratio. The third leaf of N. benthamiana (3-week old) was pressure-infiltrated with the mixed Agrobacterium suspension as described . Since the Agrobacterium infiltration into N. benthamiana leaves causes systemic spread of gene silencing signal into upper leaves and vasculature of the growing plants, the gene silencing phenotypes were observed in the newly emerged tissues at approximately 2 weeks after infiltration. The 4th leaf above the infiltrated leaf was used for semiquantitative RT-PCR.
Cloning of NbNAG
The partial NbNAG cDNA used in the VIGS screening was ~1.8 kb in length and corresponded to the N-terminal end of the predicted protein. We searched N. benthamiana and N. tabacum EST databases using Arabidopsis and rice NAG sequences to find a N. tabacum clone (BP130717) containing the C-terminal end of the NAG gene. Using a long range PCR amplification kit (Qiagen), we amplified a ~7.4 kb cDNA fragment using cDNA synthesized from N. benthamiana seedling RNA as template and primers corresponding to the 5’-termimal sequence of the ~1.8 kb cDNA (5'-caccctcaaggagagatggagaaagcag-3') and the 3’-terminal sequence of the tobacco clone (5'-agcttctgctcgacagtatccaag-3'). The amplified fragment was cloned into TOPO cloning vector (TOPOR XL PCR cloning kit) and sequenced.
GUS histochemical assay
The AtNAG promoter sequence was PCR-amplified from Arabidopsis genomic DNA using primers 5'-aagcttgtggaatattattttcaa-3' and 5'-ggatccgatcaatcgagatcgatc-3'. The 1,100 bp promoter was cloned into pBI101 vector using HindIII/BamHI sites to generate the AtNAG promoter-GUS fusion gene. The recombinant Ti-plasmid was introduced into A. tumefaciens LBA4404 for Arabidopsis transformation. GUS staining of the transgenic Arabidopsis lines was performed as described .
RNA isolation and semiquantitative RT-PCR analysis
Semiquantitative RT-PCR was performed with RNA isolated from the fourth leaf above the infiltrated leaf as described , with 15–35 cycles of amplification. The endogenous NbNAG transcript was detected using the primers 5'-ctgggagttcacctcctcca-3' and 5'-gcgagctcaacccaagaagt-3'. To detect BiP and HSP70 transcripts, the following primers were designed based on the published N. benthamiana and tobacco cDNA sequences: BI1 (5'-gcaatcgctggagttacgat-3' and 5'-ccaaggtgtgc cttctcaat-3'), BiP2 (X60059; 5'-agtgcaacagctcctgaagga-3' and ctgttacgggcatcaatcctc-3'), BiP5 (X60058; 5'-tggaagagacgcgcatccttg-3' and 5'-gaccaggatgttcttttcacc-3'), HSP70 (NP1072062; 5'-cactctcatccactgctcaga-3' and 5'-gtggtcttgtcctcagcagag-3'), and actin (5'-tggactctggtgatggtgtc-3' and 5'-cctccaatccaaacactgta-3').
Tissue sectioning, light microscopy, and transmission electron microscopy were carried out using the fourth or fifth leaf above the infiltrated leaf of the VIGS lines as described .
Measurement of in vivo H2O2, mitochondrial membrane potential, and DNA fragmentation analysis
These experiments were carried out as described .
Anti-NbNAG antibodies were generated in rabbits against an N-terminal region (351 amino acids) of NbNAG using antibody production services of AbFrontier (http://www.abfrontier.com). Preparation of BY-2 cells and immunofluorescence were performed as described [49, 50]. Fixed and permeabilized BY-2 cells were immunolabeled with 1:500 dilution of anti-NbNAG antibodies. Then the cells were incubated with 1:1000 dilution of Alexa Fluor® 594-conjugated anti-rabbit IgG antibodies (Molecular Probes). To mark the ER, the cells were briefly stained with 1 μM ER Tracker™ Blue-White DPX (Molecular Probes). Then the BY-2 cells were observed under a confocal laser scanning microscope (Carl Zeiss LSM 510) with optical filters LP 560 (excitation 543 nm, emission 560–615 nm) and BP420-480 (excitation 405 nm, emission 461 nm) for Alexa Fluor 594 and ER Tracker, respectively. The BY-2 cells were also observed under a fluorescence microscope (Olympus IX71).
Transient expression of reporter proteins
Cloning of Sporamin:GFP, Invertase:GFP, GKX, and BiP:GFP was previously described [25, 26, 40, 51]. Plasmids (15 μg) were introduced into protoplasts prepared from the fourth leaf above the infiltrated leaf of VIGS lines at 10 days after infiltration (DAI) by polyethylene glycol mediated transformation. Expression of the fusion constructs was monitored at various time points after transformation, and images were captured with a cooled CCD camera and a Zeiss Axioplan fluorescence microscope (Jena, Germany) according to .
Protein preparation and western blot analysis
To prepare cell extracts from protoplasts, transformed protoplasts were subjected to repeated freeze and thaw cycles in lysis buffer (150 mM NaCl, 20 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 3 mM MgCl2, 0.1 mg/ml antipain, 2 mg/ml aprotinins, 0.1 mg/ml E-64, 0.1 mg/ml leupeptin, 10 mg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride) and then centrifuged at 7,000 g at 4°C for 5 min . To extract proteins from culture medium, cold TCA (100 μl) was added to the medium (1 ml), and protein aggregates were precipitated by centrifugation at 10,000 g at 4°C for 5 min. The protein aggregates were dissolved in the same volume of lysis buffer used to prepare total protoplast proteins. Western blot analysis was performed using anti-GFP antibody (Clontech, Palo Alto, CA) as described previously .
Endo H treatment
Endo H treatment was performed according to . Protein extracts were prepared from transformed protoplasts and denatured in denaturation solution (1% SDS, 2% β-mercaptoethanol) by 10 min incubation at 100°C. Denatured proteins were incubated with 2 mg/ml endo H (Roche Diagnostics) in G5 buffer (50 mM sodium citrate, pH 5.5) at 37°C for 2 h. Samples were subjected to SDS-PAGE and analyzed by western blotting with anti-GFP antibody (Clontech, Palo Alto, CA).
Agrobacterium-mediated transient expression
Agro-infiltration was carried out as described . Protoplasts were prepared from the infiltrated leaves of TRV and TRV:NAG plants (15 DAI) 24 h post-infiltration, and GFP:bZIP28 fluorescence was observed by fluorescence microscopy.
Sodium 4-phenylbutyrate (PBA) treatment and ion leakage measurement
Each experiment was performed three times using 10 plants per treatment. Starting at 5 DAI, TRV:NAG plants were irrigated every other day with 1 mM PBA or distilled water until 25 DAI. Leaf discs were prepared from multiple independent TRV:NAG plants for analysis. Sample preparation and conductivity measurements were carried out as described .
Measurement of band intensity
The band intensity in the RT-PCR and immunoblotting analyses was measured using the AnalySIS LS Research program (Olympus).
Two-tailed Student’s t-tests were performed using the Minitab 16 program (Minitab Inc.; http://www.minitab.com/en-KR/default.aspx) to investigate the statistical differences between the responses of the samples. Significant differences between control and other samples were indicated by one (P ≤ 0.05) or two (P ≤0.01) asterisks.
Genbank accession number: EU602317 (NbNAG)
This research was supported by the Cooperative Research Program for Agriculture Science & Technology Development [Project numbers PJ009079 (PMBC) and PJ008214 (SSAC)] from Rural Development Administration and the Mid-career Researcher Program (No. 2012047824) from National Research Foundation of Republic of Korea.
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