The transcription factor OsNAC4 is a key positive regulator of plant hypersensitive cell death
2009; Springer Nature; Volume: 28; Issue: 7 Linguagem: Inglês
10.1038/emboj.2009.39
ISSN1460-2075
AutoresTakashi Kaneda, Yuri Taga, Ryota Takai, Megumi Iwano, Hiroyoshi Matsui, Seiji Takayama, Akira Isogai, Fang‐Sik Che,
Tópico(s)Plant Gene Expression Analysis
ResumoArticle19 February 2009free access The transcription factor OsNAC4 is a key positive regulator of plant hypersensitive cell death Takashi Kaneda Takashi Kaneda Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan Search for more papers by this author Yuri Taga Yuri Taga Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Tamura Nagahama, Shiga, Japan Search for more papers by this author Ryota Takai Ryota Takai Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan Search for more papers by this author Megumi Iwano Megumi Iwano Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan Search for more papers by this author Hiroyoshi Matsui Hiroyoshi Matsui Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Tamura Nagahama, Shiga, Japan Search for more papers by this author Seiji Takayama Seiji Takayama Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan Search for more papers by this author Akira Isogai Akira Isogai Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan Search for more papers by this author Fang-Sik Che Corresponding Author Fang-Sik Che Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Tamura Nagahama, Shiga, Japan Search for more papers by this author Takashi Kaneda Takashi Kaneda Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan Search for more papers by this author Yuri Taga Yuri Taga Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Tamura Nagahama, Shiga, Japan Search for more papers by this author Ryota Takai Ryota Takai Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan Search for more papers by this author Megumi Iwano Megumi Iwano Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan Search for more papers by this author Hiroyoshi Matsui Hiroyoshi Matsui Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Tamura Nagahama, Shiga, Japan Search for more papers by this author Seiji Takayama Seiji Takayama Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan Search for more papers by this author Akira Isogai Akira Isogai Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan Search for more papers by this author Fang-Sik Che Corresponding Author Fang-Sik Che Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Tamura Nagahama, Shiga, Japan Search for more papers by this author Author Information Takashi Kaneda1,‡, Yuri Taga2,‡, Ryota Takai1, Megumi Iwano1, Hiroyoshi Matsui2, Seiji Takayama1, Akira Isogai1 and Fang-Sik Che 1,2 1Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan 2Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Tamura Nagahama, Shiga, Japan ‡These authors contributed equally to this work *Corresponding author. Graduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266, Tamura Nagahama, Shiga 526-0829, Japan. Tel.: +81 749 64 8162; Fax: +81 749 64 8140; E-mail: [email protected] The EMBO Journal (2009)28:926-936https://doi.org/10.1038/emboj.2009.39 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The hypersensitive response (HR) is a common feature of plant immune responses and a type of programmed cell death. However, little is known about the induction mechanism of HR cell death. We report that overexpression of OsNAC4, which encodes a plant-specific transcription factor, leads to HR cell death accompanied by the loss of plasma membrane integrity, nuclear DNA fragmentation and typical morphological changes. In OsNAC4 knock-down lines, HR cell death is markedly decreased in response to avirulent bacterial strains. After induction by an avirulent pathogen recognition signal, OsNAC4 is translocated into the nucleus in a phosphorylation-dependent manner. A microarray analysis showed that the expression of 139 genes including OsHSP90 and IREN, encoding a Ca2+-dependent nuclease, were different between the OsNAC4 knock-down line and control line during HR cell death. During the induction of HR cell death, OsHSP90 is involved in the loss of plasma membrane integrity, whereas IREN causes nuclear DNA fragmentation. Overall, our results indicate that two important events occurring during HR cell death are regulated by independent pathways. Introduction Plants are continuously confronted with diverse potential pathogens; as a result, these organisms have evolved intricate immune mechanisms to recognize and defend themselves against a wide array of disease-causing agents (Jones and Dangl, 2006). One of the most efficient and immediate immune responses is hypersensitive response (HR) cell death (Lam, 2004). HR is characterized by rapid, localized death of plant cells at the site of pathogen infection (Lam, 2004). HR cell death requires active plant metabolism and intact transcriptional and translational machinery in the host plant (Richberg et al, 1998). The induction of HR is associated with signalling events, such as protein phosphorylation and the generation of reactive oxygen species (Alvarez et al, 1998). Spontaneous activation of HR cell death in the absence of pathogens has been reported in transgenic plants expressing foreign genes (Mittler et al, 1995), suggesting that HR cell death is a form of programmed cell death (PCD). HR cell death shows morphological and biochemical traits similar to animal apoptosis, the most characterized form of PCD. These common features include plasma membrane shrinkage and condensation, loss of plasma membrane integrity and nuclear DNA fragmentation resulting from inter-nucleosomal cleavage (Che et al, 1999; Tanaka et al, 2001; Yao et al, 2001). Despite these morphological and biochemical similarities, several characteristics differ between these two forms of PCD. Apoptotic bodies, which in animal cells are phagocytosed by other cells, are not formed during plant HR cell death, because of the presence of a cell wall. Whole-genome sequencing has revealed that plants lack obvious homologues to key proteins involved in animal apoptosis, such as Bcl-2 family proteins (Kutuk and Basaga, 2006) or caspases (Shi, 2002). In addition, vacuoles, which are multifunctional plant cell organelles, have an important function in plant PCD, with a key role for vacuolar processing enzyme (Hatsugai et al, 2004). Although the existence of HR cell death in plants has been recognized for many years, a description of the molecular players that serve as the central executioners has remained elusive. Acidovorax avenae is a Gram-negative bacterium that causes a seedling disease characterized by the formation of brown stripes on the sheaths of infected plants. The host range of A. avenae is wide among monocotyledonous plant; however, individual strains of the pathogen infect only one or a few host species (Kadota et al, 1996). For example, strains isolated from rice such as K1 can infect only rice plant (virulent), whereas N1141 strain isolated from finger millet cannot infect rice even after being inoculated into rice tissues (avirulent). We recently reported that an avirulent N1141, but not virulent strains such as H8301 or K1, induced immune responses in rice, including rapid cell death, oxidative burst, and transcription of several immune-related genes. This rapid cell death in rice cells induced by the avirulent strains or its flagellin, a component of the bacterial flagellum filament, was dependent on de novo protein synthesis (Che et al, 2000; Kaneda et al, 2007), accompanied by clear 180-bp nucleosomal DNA laddering and typical cell morphological changes, such as plasma membrane shrinkage and nuclear condensation (Che et al, 1999; Tanaka et al, 2001). These results suggest that the avirulent N1141 strain of A. avenae induces HR cell death in rice, which possesses all of the features that are characteristic of this process in plants. We previously identified several genes that are upregulated during HR cell death using PCR subtraction analysis and microarray analysis. Among the identified genes, the OsNAC4 gene encoding a plant-specific transcription factor (Ooka et al, 2003) was identified in both analyses. The OsNAC4 transcript was induced 3 h after incubating with the N1141 strain or its flagellin, and expression levels at 6 h after inoculation were 30-fold higher than levels before inoculation (Fujiwara et al, 2004; Kaneda et al, 2007). The activation of OsNAC4 mRNA expression did not occur when rice cells were inoculated with the strain lacking the ability to undergo HR cell death, suggesting that induction of OsNAC4 is involved in HR cell death induction (Kaneda et al, 2007). OsNAC4 has a consensus sequence known as the NAC domain (petunia NAM and Arabidopsis ATAF1 and CUC2) (Souer et al, 1996; Collinge and Boller, 2001). The finding that petunia plants with mutated NAM genes failed to form shoot apical meristems indicated that NAM plays a role in determining the position of the shoot apical meristem and primordial in this plant (Souer et al, 1996). A few NAC genes, such as AtNAC072 (RD26), AtNAC019 and AtNAC055 from Arabidopsis (Fujita et al, 2004), and BnNAC from Brassica (Hegedus et al, 2003), were found to be involved in the response to various environmental stresses. However, the exact role of OsNAC4 transcriptional factor has remained unclear. In this study, we present the induction mechanism of plant HR cell death mediated by OsNAC4. Our results show that OsNAC4 is involved in the induction of HR cell death and may control the transcription of multiple genes, including OsHSP90 and IREN. During the induction of HR cell death, Os HSP90 was involved in the loss of plasma membrane integrity, whereas IREN caused the cleavage of nuclear DNA. We propose a model in which the induction of HR cell death is regulated by a transcriptional network controlled by OsNAC4. Results Identification of OsNAC4 as HR cell death inducing transcription factor To determine the role of OsNAC4 in the induction of HR cell death in rice, we developed a transient assay in cultured rice cells based on the Arabidopsis transient assay system (Mindrinos et al, 1994). Using particle gun bombardment, two plasmids were co-introduced into cultured rice cells, one of which encoded the OsNAC4 gene and the other contained the uidA gene that encodes β-glucuronidase (GUS). Both the OsNAC4 and uidA genes were under the control of the constitutive ubiquitin promoter derived from maize (Cornejo et al, 1993). If OsNAC4 overexpression induces cell death, little GUS activity would accumulate in the transformed cultured rice cells. If overexpression does not induce cell death, transformed cells would exhibit high levels of GUS activity. As shown in Figure 1A, the same GUS activity levels were detected in both OsNAC4 plus uidA co-introduced cells (OsNAC4/uidA cells) and control uidA bombarded cells after 4 h transformation. GUS activity in OsNAC4/uidA cells was reduced by 16 h after transformation, and minimal accumulation of GUS activity was observed 24 h after transformation compared with that in control cells (Figure 1A). A similar OsNAC4-mediated reduction in GUS activity was also observed in rice leaves (Figure 1B). To confirm that the reduction in GUS activity was a measure of cell death, we introduced an OsNAC4 expression vector into protoplasts prepared from cultured rice cells, and then measured cell death using Evans blue, a marker of plasma membrane integrity. Dead protoplasts accumulated the dye, whereas live protoplasts excluded the dye (Figure 1C). After transient overexpression of OsNAC4 in rice protoplasts, almost 100% of the protoplast population was scored as dead, whereas the death rate in vector control bearing cells was as low as 10% (Figure 1D). Figure 1.Cell death induction by OsNAC4. (A) Cell death was induced in cultured rice cells by co-introduction of OsNAC4 gene (pAHC17-OsNAC4) or OsNAC6 (pAHC17-OsNAC6) and GUS. After transformation by particle bombardment, GUS activity in each cell was detected histochemically. The relative intensity of GUS staining is shown in the right graph. The mean values with error bars were derived from 10 independent experiments. (B) GUS activity in a rice leaf 24 h after transformation. GUS activity was measured after co-introduction of OsNAC4 (pAHC17-OsNAC4) and GUS (right) or GUS alone (left). pAHC17 was used for control experiments. The experiments were carried out in three different biological materials. (C) Protoplasts were transfected with the pAHC17 (control) or pAHC17-OsNAC4 vectors. After 12 h, protoplasts were incubated in 0.05% Evans blue for 5 min. (D) Protoplasts were transfected with pAHC17 (control) or pAHC17-OsNAC4. Twelve hours after transfection, dead protoplasts were scored using a light microscope. Values on the y-axis describe the percentage of dead cell in transfected protoplasts. Each determination was done with at least 1000 cells in each experiment. The mean values with error bars were derived from five independent experiments. Download figure Download PowerPoint Another important feature of HR cell death is nuclear DNA fragmentation (Che et al, 1999; Yao et al, 2001). Typically, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), which labels the free 3′-OH groups of DNA, is used to quantify DNA fragmentation and breakage (McCabe et al, 1997). Gold particles for bombardment were coated with a mixture of pAHC17-OsNAC4 and pAHC17-DsRed and then bombarded into cultured rice cells. Six hours after transformation, the OsNAC4-expressing cultured rice cells (DsRed-positive cells) exhibited fluorescein-derived bright-green fluorescence of their nuclei, as detected by TUNEL (Figure 2A–D). We constructed a DsRed-fused OsNAC4 expression vector and transformed it into cultured rice cells. TUNEL staining showed that OsNAC4-DsRed transformed cells had fluorescein-derived bright-green fluorescence signals. The positions of all bright-green signals coincided with DsRed signals (Figure 2E–H). In contrast, no bright-green fluorescence was observed in cultured rice cells transformed with DsRed alone (Figure 2I–L), indicating that OsNAC4 overexpression causes rapid DNA fragmentation accompanied with HR cell death. Figure 2.DNA fragmentation detected by TUNEL staining. (A–D) Cell images of cultured rice cells transfected with OsNAC4 and DsRed. (E–H) Cell images of cultured rice cells transfected with DsRed-fused with OsNAC4, OsNAC4-DsRed. (I–L) Cell images of cultured rice cells transfected with pAHC17 (empty vector) and DsRed. (A, E, I) are DsRed images, (B, F, J) are FITC images, (C, G, K) are DAPI images and (D, H, L) are merged images. The arrows indicate the TUNEL-positive nucleus. At least 200 transformed cells for each experiment were observed in three different biological materials. Ninety-five percent out of the OsNAC4 or OsNAC4-DsRed expressing cultured rice cells possessed TUNEL positive nuclei. Bar represents 10 μm. Download figure Download PowerPoint Suppression of HR cell death by RNA interference with OsNAC4 We selected a 501-bp region of OsNAC4 cDNA that encodes the C-terminus for RNAi experiments. This contains the 3′UTR and the TAR region, which is necessary for the transcriptional activity of OsNAC4. To determine whether this sequence is specific or shares similarity with other sequences, we searched the full-length rice cDNA database using BLASTN for short, nearly exact matches to the selected 501-bp region. No close similarity was detected for any genes other than OsNAC4, and the next level of similarity was at E=0.039. Hence, the RNAi strategy was designed to target the OsNAC4 gene specifically. We generated three lines of cultured rice cells carrying the OsNAC4 RNAi construct (NR2-2, NR2-3 and NR2-4) and one control transformant carrying the empty vector. After inoculation of the avirulent strain N1141, HR cell death measured by Evans blue staining was markedly decreased in the three RNAi transformant lines (NR2-2, NR2-3 and NR2-4) compared with the control transformant line (Figure 3A). To test whether the decrease in HR cell death correlated with reductions in OsNAC4, we quantified OsNAC4 protein levels in all transformant lines using an anti-OsNAC4 antibody. Immunoblot analysis of wild-type cell extracts using an anti-OsNAC4 antibody specifically detected a protein migrating with an apparent molecular mass of 35 kDa (Figure 3B; Supplementary Figure S1). The cell lines with decreased HR cell death, NR2-2, NR2-3 and NR2-4, exhibited 10-fold or greater reductions in OsNAC4 protein levels. In addition, the level of reduction in HR cell death was strongly associated with a reduction in OsNAC4 protein accumulation (Figure 3B). Figure 3.Suppression of HR cell death in OsNAC4 RNAi transformants. (A) Cell death detected by Evans blue staining in OsNAC4 RNAi transformant lines (NR2-2, NR2-3 and NR2-4) before (solid column) and 8 h after (open column) inoculation with the avirulent N1141 strain. (B) Accumulation of OsNAC4 was detected with an anti-OsNAC4 antibody in OsNAC4 RNAi knock-down lines. Proteins (10 μg) from vector transformed cells (Cont) and RNAi transformant lines were separated on a 12.5% (w/v) SDS–PAGE and transferred to a nitrocellulose membrane. Then OsNAC4 were detected by immunoblotting with an anti-OsNAC4 antibody (top part). The same amount of each fraction was separated by SDS–PAGE and proteins were detected by silver staining (lower part). (C) DNA fragmentation in OsNAC4 knock-down (left panels) and control (right panels) lines was assessed after inoculation with the avirulent N1141 strain. TUNEL (upper panels) and DAPI (lower panels) staining are shown. Bar represents 10 μm. (D) Percentage of TUNEL-positive nuclei in NR2-4 and vector transformed lines (control line) inoculated with N1141. The percentage of TUNEL-positive nuclei was determined by counting nuclei within 20 individual fields. Each determination was done with at least 2000 nuclei in each of three independent experiments. A full colour version of this figure is available at The EMBO Journal online. Download figure Download PowerPoint We evaluated DNA fragmentation in OsNAC4 knock-down and control lines after inoculation with the avirulent N1141 strain. TUNEL staining of NR2-4 RNAi transformants did not produce fluorescent nuclei (Figure 3C), whereas control cells exhibited multiple intensely stained nuclei 12 h after inoculation (Figure 3C). We also examined the percentage of TUNEL-positive nuclei in the above experiment. In the control line inoculated with the avirulent strain, approximately 19% of nuclei had FITC-derived fluorescence 12 h after inoculation (Figure 3D). In contrast, approximately 2% of TUNEL-positive nuclei were detected in the N1141-inoculated NR2-4 line (Figure 3D). We next studied the ultrastructure of NR2-4 and control cells undergoing the induction of HR cell death using transmission electron microscopy. Mock-inoculated cells contained regularly shaped nuclei with an intact nuclear envelope (Figure 4A), and the cytoplasm showed a highly ordered structure (Figure 4D). Twelve hours after inoculation of the avirulent strain, control cells had nuclei undergoing HR cell death that exhibited an irregular shape with invaginations and an electron-dense structure that may have resulted from chromatin condensation (Figure 4B). The plasma membrane also appeared to have partially separated from the cell wall (Figure 4E). In contrast, these morphological changes in the nucleus and plasma membrane were not observed when the OsNAC4-deficient line NR2-4 was inoculated with the avirulent strain (Figure 4C and F). From these observations, we concluded that OsNAC4 is a key transcription factor regulating the induction of HR cell death in rice. Figure 4.Morphological analysis using transmission electron microscopy. (A) A nucleus in a cell transformed with empty vector (control cell) 12 h after water treatment. The arrow indicates a normal nucleus. (B) A nucleus in empty vector-transformed cells (control cell) 12 h after inoculation with the avirulent N1141 strain. The arrow indicates condensed nucleus. (C) A nucleus in the OsNAC4 knock-down NR2-4 line after a 12-h inoculation with the avirulent N1141 strain. The arrow indicates a normal nucleus. (D) The cell wall and plasma membrane in a cell transformed with empty vector (control cell) 12 h after water treatment. The arrow indicates a normal plasma membrane. (E) The cell wall and plasma membrane in empty vector transformed cells (control cell) 12 h after inoculation with the avirulent N1141 strain. The arrows indicate the separated-plasma membrane from the cell wall. (F) The cell wall and plasma membrane in the OsNAC4 knock-down NR2-4 line after a 12-h inoculation with the avirulent N1141 strain. The arrow indicates a normal plasma membrane. CW, cell wall; NC, nucleus; NM, nuclear membrane; PM, plasma membrane. Bar represents 1 μm. Download figure Download PowerPoint Translocation of OsNAC4 into the nucleus depends on phosphorylation As H2O2 is known as an important signalling molecule for HR induction, we analyzed the generation of H2O2 in OsNAC4-RNAi cultured cells. When the incompatible flagellin was added to cultured rice cells, rapid H2O2 generation was observed (Supplementary Figure S2). The same generation pattern of H2O2 was also observed in OsNAC4-RNAi NR2-4 cells even when HR cell death was abolished (Supplementary Figure S2), suggesting that OsNAC4 is located down-stream of H2O2 signals in the HR induction pathway. We next examined whether induction of OsNAC4-mediated cell death in cultured rice cells is involved in protein phosphorylation signalling. For this purpose, staurosporine, a potent inhibitor of plant and animal serine/threonine protein kinases, was used. In the absence of staurosporine, cell death induced by the avirulent N1141 strain was detected by Evans blue staining 6 h after inoculation, and the number of dead cells gradually increased. Conversely, HR cell death was completely blocked by staurosporine treatment (2 μM) for up to 9 h after inoculation (Figure 5A). During this experimental period, bacterial growth rates were the same in the presence and absence of 2 μM staurosporine. Figure 5.Effects of protein phosphorylation on OsNAC4-mediated cell death. (A) Time-course of cell death in cultured rice cells after inoculation with the avirulent N1141 strain in the presence of 2 μM staurosporine (open circles) or absence of the inhibitor (solid circles). The amount of cell death was estimated by Evans blue staining of individual cells at 595 nm. Each data point represents the average of three independent experiments. Bars indicate the standard errors. (B) Cell death in protoplasts transfected with the pAHC17 (control) and pAHC17-OsNAC4 vectors in the presence or absence of 2 μM staurosporine. Protoplasts were treated with staurosporine concurrent with transfection. Twelve hours after transfection, dead protoplasts were scored using a light microscope. Y-axis values represent the percentage of transfected protoplasts that were determined to be dead. Each data point represents the average of three independent experiments. Bars indicate the standard errors. (C) Time-dependent accumulation of OsNAC4 mRNA in cultured rice cells after inoculation with the avirulent N1141 strain of A. avenae in the presence (open circles) or absence (closed circles) of 2 μM staurosporine. The amount of each mRNA was measured by real-time RT–PCR; these values were calculated from the threshold point located in the log-linear range of RT–PCR. Quantification of each OsNAC4 mRNA was calculated with calibration curve, which was prepared using standard OsNAC4 genes of known template amounts and corrected with reference data of Act-1 gene. Each data point represents the average of three independent experiments. Bars indicate the standard errors. Download figure Download PowerPoint To clarify whether OsNAC4-mediated HR cell death is also inhibited by staurosporine, protoplasts overexpressing OsNAC4 were treated with staurosporine and then examined for the inhibition of cell death by Evans blue. A large proportion of dead cells were observed in cells overexpressing OsNAC4; staurosporine reduced the amount of cell death (Figure 5B). To study the step in OsNAC4-induced cell death that required protein phosphorylation, we examined the effects of staurosporine on OsNAC4 mRNA expression using quantitative real-time RT–PCR in cultured rice cells inoculated with the avirulent N1141 strain. The OsNAC4 induction kinetics after inoculation with the N1141 strain was unaffected by the addition of staurosporine (Figure 5C), suggesting that inhibition of protein phosphorylation does not influence the induction of OsNAC4 mRNA during HR cell death. We next examined the subcellular localization of OsNAC4 during HR cell death induction by immunoblot analysis. Inoculation with the avirulent strain increased the amount of endogenous OsNAC4 protein in the nuclear fraction in a time-dependent manner, whereas treatment with water alone did not affect the proportion of OsNAC4 in the nuclear fraction (Figure 6A). The amount of OsNAC4 within the cytosolic fraction was slightly decreased by avirulent N1141 inoculation (Figure 6A). Moreover, the total amount of OsNAC4 protein in whole cells was slightly increased during HR cell death induction (data not shown). We also examined the nuclear translocation of OsNAC4 in cultured rice cells inoculated with the virulent K1 strain. Western blot analysis showed that OsNAC4 does not translocate into nuclei during the compatible interactions (data not shown). Figure 6.Translocation of OsNAC4 into the nucleus and phosphorylation of OsNAC4 during HR cell death. (A) Time-dependent accumulation of OsNAC4 in cultured rice cells after inoculation with the avirulent N1141 strain. The upper panels represent the amount of OsNAC4 protein in the nuclear fraction. Nuclear fractions (10 μg protein) were separated by SDS–PAGE and transferred to a nitrocellulose membrane. Then OsNAC4 were detected by immunoblotting with an anti-OsNAC4 antibody (top part). The same amount of each nuclear fraction was separated by SDS–PAGE and proteins were detected by silver staining (middle part). The lower panel represents the proportion of OsNAC4 in the cytosolic fraction. Total protein (10 μg) from each fraction was separated by SDS–PAGE and transferred to a nitrocellulose membrane. Then OsNAC4 were detected by immunoblotting with an anti-OsNAC4 antibody (upper part). The same amount of each cytosolic fraction was separated by SDS–PAGE and proteins were detected by silver staining (lower part). The purity of each fraction was analysed by immunoblotting using an antihistone H3 antibody (nucleus-specific antibody) and an anti-OsUSP (cytosol-specific antibody). (B) Immunogold labelling of OsNAC4 using anti-OsNAC4 antibody. A control cell line (upper panels) and the NR2-4 OsNAC4 knock-down line (middle panels) were visualized 0 h (left panels) and 12 h (right panels) after inoculation with the avirulent N1141 strain. A control cells treated with preimmune serum were visualized 0 h (left panels) and 12 h (right panels) after inoculation with the N1141 strain (lower panels). Bar represents 1 μm. (C) The number of gold particles in the nucleus and cytosol when the OsNAC4 protein was immunogold labelled. The number of gold particles in nucleus and cytosol were visually counted. Pre-IS, preimmune serum; N, nucleus; C, cytosol. Each data represent the average of the particle number in five counted area (1 μm2). Bars indicate the standard errors. (D) The localization of NES-fused OsNAC4-DsRed and OsNAC4-DsRed after 6 h transformation in rice protoplasts. NES fused to the N-terminal of OsNAC4-DsRed (left panel) and OsNAC4-DsRed (right panel). Bar represents 10 μm. (E) Cell death in protoplasts transfected with the NES-OsNAC4-DsRed and OsNAC4-DsRed (control) vectors. Six hours after transfection, dead protoplasts were scored using a light microscope. Y-axis values represent the percentage of transfected protoplasts that were determined to be dead. Each determination was done with at least 200 cells in each of three independent experiments. Bars indicate the standard errors. (F) The time-dependent accumulation of OsNAC4 in nuclei isolated from cultured rice cells after inoculation with the avirulent N1141 strain. The left three lanes represent nuclear OsNAC4 in water-treated cells, whereas the right three lanes represent OsNAC4 in cells treated with 2 μM staurosporine. Total protein (10 μg) from each fraction was separated by SDS–PAGE and transferred to a nitrocellulose membrane
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