Light Activates the Translational Regulatory Kinase GCN2 via Reactive Oxygen Species Emanating from the Chloroplast.

Cytosolic mRNA translation is subject to global and mRNA-specific controls. Phosphorylation of the translation initiation factor eIF2α anchors a reversible regulatory switch that represses cytosolic translation globally. The stress-responsive GCN2 kinase is the only known kinase for eIF2α serine 56 in Arabidopsis (Arabidopsis thaliana). Here, we show that conditions that generate reactive oxygen species (ROS) in the chloroplast, including dark-light transitions, high light, and the herbicide methyl viologen, rapidly activated GCN2 kinase, whereas mitochondrial and endoplasmic reticulum stress did not. GCN2 activation was light dependent and mitigated by photosynthesis inhibitors and ROS quenchers. Accordingly, the seedling growth of multiple Arabidopsis gcn2 mutants was retarded under excess light conditions, implicating the GCN2-eIF2α pathway in responses to light and associated ROS. Once activated, GCN2 kinase preferentially suppressed the ribosome loading of mRNAs for functions such as mitochondrial ATP synthesis, the chloroplast thylakoids, vesicle trafficking, and translation. The gcn2 mutant overaccumulated transcripts functionally related to abiotic stress, including oxidative stress, as well as innate immune responses. Accordingly, gcn2 displayed defects in immune priming by the fungal elicitor, chitin. Therefore, we provide evidence that reactive oxygen species produced by the photosynthetic apparatus help activate the highly conserved GCN2 kinase, leading to eIF2α phosphorylation and thus affecting the status of the cytosolic protein synthesis apparatus.

TaADF4, an actin-depolymerizing factor from wheat, is required for resistance to the stripe rust pathogen Puccinia striiformis f. sp. tritici.

Actin filament assembly in plants is a dynamic process, requiring the activity of more than 75 actin-binding proteins. Central to the regulation of filament assembly and stability is the activity of a conserved family of actin-depolymerizing factors (ADFs), whose primarily function is to regulate the severing and depolymerization of actin filaments. In recent years, the activity of ADF proteins has been linked to a variety of cellular processes, including those associated with response to stress. Herein, a wheat ADF gene, TaADF4, was identified and characterized. TaADF4 encodes a 139-amino-acid protein containing five F-actin-binding sites and two G-actin-binding sites, and interacts with wheat (Triticum aestivum) Actin1 (TaACT1), in planta. Following treatment of wheat, separately, with jasmonic acid, abscisic acid or with the avirulent race, CYR23, of the stripe rust pathogen Puccinia striiformis f. sp. tritici, we observed a rapid induction in accumulation of TaADF4 mRNA. Interestingly, accumulation of TaADF4 mRNA was diminished in response to inoculation with a virulent race, CYR31. Silencing of TaADF4 resulted in enhanced susceptibility to CYR23, demonstrating a role for TaADF4 in defense signaling. Using a pharmacological-based approach, coupled with an analysis of host response to pathogen infection, we observed that treatment of plants with the actin-modifying agent latrunculin B enhanced resistance to CYR23, including increased production of reactive oxygen species and enhancement of localized hypersensitive cell death. Taken together, these data support the hypothesis that TaADF4 positively modulates plant immunity in wheat via the modulation of actin cytoskeletal organization.

The Pseudomonas syringae Type III Effector HopG1 Induces Actin Remodeling to Promote Symptom Development and Susceptibility during Infection.

The plant cytoskeleton underpins the function of a multitude of cellular mechanisms, including those associated with developmental- and stress-associated signaling processes. In recent years, the actin cytoskeleton has been demonstrated to play a key role in plant immune signaling, including a recent demonstration that pathogens target actin filaments to block plant defense and immunity. Herein, we quantified spatial changes in host actin filament organization after infection with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), demonstrating that the type-III effector HopG1 is required for pathogen-induced changes to actin filament architecture and host disease symptom development during infection. Using a suite of pathogen effector deletion constructs, coupled with high-resolution microscopy, we found that deletion of hopG1 from Pst DC3000 resulted in a reduction in actin bundling and a concomitant increase in the density of filament arrays in Arabidopsis, both of which correlate with host disease symptom development. As a mechanism underpinning this activity, we further show that the HopG1 effector interacts with an Arabidopsis mitochondrial-localized kinesin motor protein. Kinesin mutant plants show reduced disease symptoms after pathogen infection, which can be complemented by actin-modifying agents. In total, our results support a model in which HopG1 induces changes in the organization of the actin cytoskeleton as part of its virulence function in promoting disease symptom development.

Development of disease-resistant rice by optimized expression of WRKY45.

The rice transcription factor WRKY45 plays a central role in the salicylic acid signalling pathway and mediates chemical-induced resistance to multiple pathogens, including Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae. Previously, we reported that rice transformants overexpressing WRKY45 driven by the maize ubiquitin promoter were strongly resistant to both pathogens; however, their growth and yield were negatively affected because of the trade-off between the two conflicting traits. Also, some unknown environmental factor(s) exacerbated this problem. Here, we report the development of transgenic rice lines resistant to both pathogens and with agronomic traits almost comparable to those of wild-type rice. This was achieved by optimizing the promoter driving WRKY45 expression. We isolated 16 constitutive promoters from rice genomic DNA and tested their ability to drive WRKY45 expression. Comparisons among different transformant lines showed that, overall, the strength of WRKY45 expression was positively correlated with disease resistance and negatively correlated with agronomic traits. We conducted field trials to evaluate the growth of transgenic and control lines. The agronomic traits of two lines expressing WRKY45 driven by the OsUbi7 promoter (PO sUbi7 lines) were nearly comparable to those of untransformed rice, and both lines were pathogen resistant. Interestingly, excessive WRKY45 expression rendered rice plants sensitive to low temperature and salinity, and stress sensitivity was correlated with the induction of defence genes by these stresses. These negative effects were barely observed in the PO sUbi7 lines. Moreover, their patterns of defence gene expression were similar to those in plants primed by chemical defence inducers.

The plant actin cytoskeleton responds to signals from microbe-associated molecular patterns.

Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.

WRKY45-dependent priming of diterpenoid phytoalexin biosynthesis in rice and the role of cytokinin in triggering the reaction.

Plant activators such as benzothiadiazole (BTH) protect plants against diseases by priming the salicylic acid (SA) signaling pathway. In rice, the transcription factor WRKY45 plays a central role in this process. To investigate the mechanism involved in defense-priming by BTH and the role of WRKY45 in this process, we analyzed the transcripts of biosynthetic genes for diterpenoid phytoalexins (DPs) during the rice-Magnaporthe oryzae interaction. The DP biosynthetic genes were barely upregulated in BTH-treated rice plants, but were induced rapidly after M. oryzae infection in a WRKY45-dependent manner. These results indicate that the DP biosynthetic genes were primed by BTH through WRKY45. Rapid induction of the DP biosynthetic genes was also observed after M. oryzae infection to WRKY45-overexpressing (WRKY45-ox) plants. The changes in gene transcription resulted in accumulation of DPs in WRKY45-ox and BTH-pretreated rice after M. oryzae infection. Previously, we reported that cytokinins (CKs), especially isopentenyladenines, accumulated in M. oryzae-infected rice. Here, we show that DP biosynthetic genes are regulated by the SA/CK synergism in a WRKY45-dependent manner. Together, we propose that CK plays a role in mediating the signal of M. oryzae infection to trigger the induction of DP biosynthetic genes in BTH-primed plants.

Arabidopsis Actin-Depolymerizing Factor-4 links pathogen perception, defense activation and transcription to cytoskeletal dynamics.

The primary role of Actin-Depolymerizing Factors (ADFs) is to sever filamentous actin, generating pointed ends, which in turn are incorporated into newly formed filaments, thus supporting stochastic actin dynamics. Arabidopsis ADF4 was recently shown to be required for the activation of resistance in Arabidopsis following infection with the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst) expressing the effector protein AvrPphB. Herein, we demonstrate that the expression of RPS5, the cognate resistance protein of AvrPphB, was dramatically reduced in the adf4 mutant, suggesting a link between actin cytoskeletal dynamics and the transcriptional regulation of R-protein activation. By examining the PTI (PAMP Triggered Immunity) response in the adf4 mutant when challenged with Pst expressing AvrPphB, we observed a significant reduction in the expression of the PTI-specific target gene FRK1 (Flg22-Induced Receptor Kinase 1). These data are in agreement with recent observations demonstrating a requirement for RPS5 in PTI-signaling in the presence of AvrPphB. Furthermore, MAPK (Mitogen-Activated Protein Kinase)-signaling was significantly reduced in the adf4 mutant, while no such reduction was observed in the rps5-1 point mutation under similar conditions. Isoelectric focusing confirmed phosphorylation of ADF4 at serine-6, and additional in planta analyses of ADF4's role in immune signaling demonstrates that nuclear localization is phosphorylation independent, while localization to the actin cytoskeleton is linked to ADF4 phosphorylation. Taken together, these data suggest a novel role for ADF4 in controlling gene-for-gene resistance activation, as well as MAPK-signaling, via the coordinated regulation of actin cytoskeletal dynamics and R-gene transcription.

Expression and localization of aqua-glyceroporins AQP3 and AQP9 in rat oral epithelia.

Aquaporins (AQPs) are a family of small integral membrane proteins made up of 6 hydrophobic, a-helical, membrane-spanning domains surrounding a highly selective aqueous pore. AQP3, AQP7, and AQP9, termed aqua-glyceroporins, are known to be involved in the transport of water, glycerol, and other small molecules. In this study, we investigated the expression and localization of aqua-glyceroporins in rat oral stratified squamous epithelia of the palate, the buccal mucosa, the inferior aspect of the tongue, and the oral floor by using RT-PCR, immunofluorescence, and immunogold electron microscopy. AQP3 and AQP9 mRNAs were expressed in whole oral epithelium. Immunostaining for AQP3 was recognized in each type of epithelium. The results suggest that AQP3 synthesis begins predominantly in the cytoplasm of the basal cells. During the process of epithelial cell differentiation, AQP3 protein appears to accumulate and be transported to the plasma membrane, from where it is incorporated into the cornified or surface layers. The intracellular localization of AQP3 appears to correlate with the differentiation of keratinocytes, suggesting that it acts as an enhancer of the physiological permeability barrier together with membrane coating granules. The distribution pattern of AQP9 was limited to the marginal areas of the basal and suprabasal layers, which was different from that of AQP3. This difference in distribution between AQP3 and AQP9 suggests that AQP9 in rat oral epithelia acts as a channel by facilitating glycerol uptake from the blood through the endothelial cells of the capillary vessels to the oral stratified squamous epithelium. AQP3 and AQP9 facilitate both transcellular osmotic water flow and glycerol transport as pore-like passive transporters in the keratinocytes of oral epithelia, and may play a key role in not only hydration and the permeability barrier, but also cell proliferation, differentiation, migration, development, and wound healing by generating ATP.

Cytokinins act synergistically with salicylic acid to activate defense gene expression in rice.

Hormone crosstalk is pivotal in plant-pathogen interactions. Here, we report on the accumulation of cytokinins (CK) in rice seedlings after infection of blast fungus Magnaporthe oryzae and its potential significance in rice-M. oryzae interaction. Blast infection to rice seedlings increased levels of N(6)-(Δ(2)-isopentenyl) adenine (iP), iP riboside (iPR), and iPR 5'-phosphates (iPRP) in leaf blades. Consistent with this, CK signaling was activated around the infection sites, as shown by histochemical staining for ß-glucuronidase activity driven by a CK-responsive OsRR6 promoter. Diverse CK species were also detected in the hyphae (mycelium), conidia, and culture filtrates of blast fungus, indicating that M. oryzae is capable of production as well as hyphal secretion of CK. Co-treatment of leaf blades with CK and salicylic acid (SA), but not with either one alone, markedly induced pathogenesis-related genes OsPR1b and probenazole-induced protein 1 (PBZ1). These effects were diminished by RNAi-knockdown of OsNPR1 or WRKY45, the key regulators of the SA signaling pathway in rice, indicating that the effects of CK depend on these two regulators. Taken together, our data imply a coevolutionary rice-M. oryzae interaction, wherein M. oryzae probably elevates rice CK levels for its own benefits such as nutrient translocation. Rice plants, on the other hand, sense it as an infection signal and activate defense reactions through the synergistic action with SA.

Genome-wide identification of WRKY45-regulated genes that mediate benzothiadiazole-induced defense responses in rice.

BACKGROUND: The rice transcription factor WRKY45 plays a crucial role in salicylic acid (SA)/benzothiadiazole (BTH)-induced disease resistance. Its knockdown severely reduces BTH-induced resistance to the fungal pathogen Magnaporthe oryzae and the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). Conversely, overexpression of WRKY45 induces extremely strong resistance to both of these pathogens. To elucidate the molecular basis of WRKY45-dependent disease resistance, we analyzed WRKY45-regulated gene expression using rice transformants and a transient gene expression system. RESULTS: We conducted a microarray analysis using WRKY45-knockdown (WRKY45-kd) rice plants, and identified WRKY45-dependent genes among the BTH-responsive genes. The BTH-responsiveness of 260 genes was dependent on WRKY45. Among these, 220 genes (85%), many of which encoded PR proteins and proteins associated with secondary metabolism, were upregulated by BTH. Only a small portion of these genes overlapped with those regulated by OsNPR1/NH1, supporting the idea that the rice SA pathway branches into WRKY45- regulated and OsNPR1/NH1-regulated subpathways. Dexamethazone-induced expression of myc-tagged WRKY45 in rice immediately upregulated transcription of endogenous WRKY45 and genes encoding the transcription factors WRKY62, OsNAC4, and HSF1, all of which have been reported to have defense-related functions. This was followed by upregulation of defense genes encoding PR proteins and secondary metabolic enzymes. Many of these genes were also induced after M. oryzae infection. Their temporal transcription patterns were consistent with those after dexamethazone-induced WRKY45 expression. In a transient expression system consisting of particle bombardment of rice coleoptiles, WRKY45 acted as an effector to trans-activate reporter genes in which the luciferase coding sequence was fused to upstream and intragenic sequences of WRKY62 and OsNAC4. Trans-activation of transcription occurred through a W-box-containing sequence upstream of OsNAC4 and mutations in the W-boxes abolished the trans-activation. CONCLUSIONS: These data suggest a role of WRKY45 in BTH-induced disease resistance as a master regulator of the transcriptional cascade regulating defense responses in one of two branches in the rice SA pathway.

WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance.

OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice cells. Overexpression of OsWRKY76 in rice plants resulted in drastically increased susceptibility to M. oryzae, but improved tolerance to cold stress. Microarray analysis revealed that overexpression of OsWRKY76 suppresses the induction of a specific set of PR genes and of genes involved in phytoalexin synthesis after inoculation with blast fungus, consistent with the observation that the levels of phytoalexins in the transgenic rice plants remained significantly lower than those in non-transformed control plants. Furthermore, overexpression of OsWRKY76 led to the increased expression of abiotic stress-associated genes such as peroxidase and lipid metabolism genes. These results strongly suggest that OsWRKY76 plays dual and opposing roles in blast disease resistance and cold tolerance.

Mapping and characterization of seed dormancy QTLs using chromosome segment substitution lines in rice.

Seed dormancy--the temporary failure of a viable seed to germinate under favorable conditions--is a complex characteristic influenced by many genes and environmental factors. To detect the genetic factors associated with seed dormancy in rice, we conducted a QTL analysis using chromosome segment substitution lines (CSSLs) derived from a cross between Nona Bokra (strong dormancy) and Koshihikari (weak dormancy). Comparison of the levels of seed dormancy of the CSSLs and their recurrent parent Koshihikari revealed that two chromosomal regions-on the short arms of chromosomes 1 and 6-were involved in the variation in seed dormancy. Further genetic analyses using an F(2) population derived from crosses between the CSSLs and Koshihikari confirmed the allelic differences and the chromosomal locations of three putative QTLs: Sdr6 on chromosome 1 and Sdr9 and Sdr10 on chromosome 6. The Nona Bokra alleles of the three QTLs were associated with decreased germination rate. We discuss the physiological features of the CSSLs and speculate on the possible mechanisms of dormancy in light of the newly detected QTLs.

Scanning and transmission electron microscopic observation of changes in cylindrical cytoplasmic processes of isolated single merkel cell.

The aim of the present study was to determine the reason isolated single Merkel cells do not respond to mechanical stimulation by fluorescent or histological techniques. Cells identified as Merkel cells by quinacrine fluorescence and measurement of intracellular calcium concentration were observed by transmission electron and scanning electron microscopy. Observations elucidated that the cylindrical cytoplasmic processes of single Merkel cells disappeared with time shortly after isolation. Transmission electron microscopy revealed the presence of numerous dense-cored granules, which may function as sensory receptors in the cytoplasm of the isolated single Merkel cell. Disappearance of the cylindrical cytoplasmic processes impeded reception of mechanical stimulation. The results suggest that an isolated single Merkel cell continues to function as a sensory receptor cell due to the presence of numerous dense-cored granules. Furthermore, the results show that an isolated single Merkel cell is not an appropriate specimen for investigation of mechanically-gated channels.

Effect of diode laser on proliferation and differentiation of PC12 cells.

This study investigated the effects of diode (GaAlAs) laser irradiation at an effective energy density of 5 or 20 J/cm(2) on cell growth factor-induced differentiation and proliferation in pheochromocytoma cells (PC12 cells), and whether those effects were related to activation of the p38 pathway. Laser irradiation at 20 J/cm(2) significantly decreased the number of PC12 cells, while no difference was seen between the 5 J/cm(2) group and the control group (p<0.05). Western blotting revealed marked expression of neurofilament and ß-tubulin, indicating greater neurite differentiation in the irradiation groups than in the control group at 48 hr. Irradiation also enhanced expression of phospho-p38. The decrease in number of cells after laser irradiation was accelerated by p38 inhibitor, while neurite differentiation was up-regulated by laser irradiation, even when the p38 pathway was blocked. This suggests that laser irradiation up-regulated neurite differentiation in PC12 cells involving p38 and another pathway.

Epithelial cell rests of Malassez modulate cell proliferation, differentiation and apoptosis via gap junctional communication under mechanical stretching in vitro.

Epithelial cell rests of Malassez (ERM) are involved in the maintenance and homeostasis of the periodontal ligament. The objective of this study was to investigate the effect of mechanical stretching on cell growth, cell death and differentiation in the ERM. Cultured porcine ERM were stretched for 24 hr in cycles of 18% elongation for 1 sec followed by 1 sec relaxation. The numbers of cells and TUNEL-positive cells were then counted. The expression of mRNAs encoding gap junction protein α1 (Gja1), ameloblastin, bone morphogenetic protein 2 (BMP2), bone morphogenetic protein 4 (BMP4) and noggin were evaluated using quantitative real-time PCR. The number of cells in the stretching group was approximately 1.3-fold higher than that in the non-stretching controls at 24 hr (p<0.01). Apoptotic cells ranged from 1.9-2.5% in the stretching group at 24 hr, but were only 0.6% in the control group (p<0.01). The expression of Gja1, ameloblastin and noggin mRNAs in the stretching group was decreased at 24 hr compared with in the non-stretching group (p<0.01), whereas the expression of BMP2 and BMP4 mRNAs in the stretching group was significantly higher than that in the control group (p<0.01). Incorporation of 18 α-glycyrrhetinic acid (18GA, a gap junction inhibitor) promoted proliferation and apoptosis and confirmed both the increase of BMP2 and BMP4 and the decline of Gja1, ameloblastin and noggin in ERM. Thus, the ERM modulate cell proliferation and apoptosis, and inhibit differentiation by reducing expression of Gja1 under mechanical stretching.

Osteosarcoma of maxilla with unusual image findings in child.

Osteosarcoma of the head and neck is relatively rare and accounts for less than 10 percent of all osteosarcomas in general. We report a case of osteosarcoma in which imaging and histopathology of the hard palate of an 11-year-old boy yielded atypical findings. An approximately 8×15mm lesion found in the center of the palate was hard and healthy in color. Subsequent biopsy resulted in a diagnosis of nonepithelial malignant tumor. No abnormalities were observed in the maxillary bone or tooth on panoramic or occlusal radiographs. Computed tomography images revealed a mass lesion approximately 7×9×9mm in size on the hard palate extending into the maxilla. The cortex of the maxilla adjacent to the lesion was unclear in parts. The internal structures were slightly inhomogeneous and its density was lower than that of muscle. On magnetic resonance images, the lesion was represented by low signal intensity on T1-weighted (T1W) images and high signal intensity on T2-weighted images with fat-suppression. The margin of the lesion was a little unclear and the internal structures were slightly inhomogeneous. The lesion was enhanced homogeneously on post-contrast T1W images with fat-suppression. The histopathological diagnosis was fibrogenesis-type osteosarcoma. No findings specific to osteosarcoma such as localized enlargement of the periodontal ligament space alongside the root, cortical destruction, periosteal ossification or osteogenesis were found in this case.

Abscisic acid interacts antagonistically with salicylic acid signaling pathway in rice-Magnaporthe grisea interaction.

Plant hormones play pivotal signaling roles in plant-pathogen interactions. Here, we report characterization of an antagonistic interaction of abscisic acid (ABA) with salicylic acid (SA) signaling pathways in the rice-Magnaporthe grisea interaction. Exogenous application of ABA drastically compromised the rice resistance to both compatible and incompatible M. grisea strains, indicating that ABA negatively regulates both basal and resistance gene-mediated blast resistance. ABA markedly suppressed the transcriptional upregulation of WRKY45 and OsNPR1, the two key components of the SA signaling pathway in rice, induced by SA or benzothiadiazole or by blast infection. Overexpression of OsNPR1 or WRKY45 largely negated the enhancement of blast susceptibility by ABA, suggesting that ABA acts upstream of WRKY45 and OsNPR1 in the rice SA pathway. ABA-responsive genes were induced during blast infection in a pattern reciprocal to those of WRKY45 and OsPR1b in the compatible rice-blast interaction but only marginally in the incompatible one. These results suggest that the balance of SA and ABA signaling is an important determinant for the outcome of the rice-M. grisea interaction. ABA was detected in hyphae and conidia of M. grisea as well as in culture media, implying that blast-fungus-derived ABA could play a role in triggering ABA signaling at host infection sites.

Role of OsNPR1 in rice defense program as revealed by genome-wide expression analysis.

NPR1 is a central regulator of salicylic-acid (SA)-mediated defense signaling in Arabidopsis. Here, we report the characterization of OsNPR1, an Oryzae sativa (rice) ortholog of NPR1, focusing on its role in blast disease resistance and identification of OsNPR1-regulated genes. Blast resistance tests using OsNPR1 knockdown and overexpressing rice lines demonstrated the essential role of OsNPR1 in benzothiadiazole (BTH)-induced blast resistance. Genome-wide transcript profiling using OsNPR1-knockdown lines revealed that 358 genes out of 1,228 BTH-upregulated genes and 724 genes out of 1,069 BTH-downregulated genes were OsNPR1-dependent with respect to BTH responsiveness, thereby indicating that OsNPR1 plays a more vital role in gene downregulation. The OsNPR1-dependently downregulated genes included many of those involved in photosynthesis and in chloroplast translation and transcription. Reduction of photosynthetic activity after BTH treatment and its negation by OsNPR1 knockdown were indeed reflected in the changes in Fv/Fm values in leaves. These results imply the role of OsNPR1 in the reallocation of energy and resources during defense responses. We also examined the OsNPR1-dependence of SA-mediated suppression of ABA-induced genes.

Sodium-calcium exchangers in rat ameloblasts.

Although the central role of ameloblasts in synthesis and resorption of enamel matrix proteins during amelogenesis is well documented, the Ca(2+)-transport/extrusion mechanism remains to be fully elucidated. To clarify Ca(2+)-transport in rat ameloblasts, we investigated expression and localization of Na(+)-Ca(2+) exchanger (NCX) isoforms and the functional characteristics of their ion transporting/pharmacological properties. RT-PCR and immunohistochemical analyses revealed expression of NCX1 and NCX3 in ameloblasts, localized in the apical membrane. In patch-clamp recordings, Ca(2+) efflux by Na(+)-Ca(2+) exchange showed dependence on external Na(+). Ca(2+) influx by Na(+)-Ca(2+) exchange, measured by fura-2 fluorescence, showed dependence on extracellular Ca(2+) concentration, and it was blocked by NCX inhibitors KB-R7943, SEA0400, and SN-6. These results showed significant expression of NCX1 and NCX3 in ameloblasts, indicating their involvement in the directional Ca(2+) extrusion pathway from cells to the enamel mineralizing front.

Effect of stretching stress on gene transcription related to early-phase differentiation in rat periodontal ligament cells.

Mechanical stress such as occlusal and orthodontic loading has been suggested to induce a homeostatic and regenerative response in periodontal ligament (PDL), but the underlying mechanism remains to be clarified. The purpose of this study was to investigate expression of mRNAs encoding proteins involved in osteogenesis and homeostasis by PDL cells following application of tensile stress and characterize the relationship between such expression and the regenerative and homeostatic functions of the PDL. PDL cells were obtained from rats and stretched by 9% or 18% at a frequency of 6 cycles/min for 12 hr to 5 days in a FX-4000T™ culture system. After stretching, expression of mRNAs encoding collagen type I (Col-I), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-4 (BMP-4), heat shock protein 70 (HSP70) and basic fibroblast growth factor (bFGF) was investigated. The highest levels of Col-I, ALP and BMP-2 mRNA expression occurred at 12 hr, while those of BMP-4 and HSP70 occurred at 1 day and 5 days, respectively. Expression levels of Col-I, ALP, BMP-2, BMP-4 and HSP70 increased magnitude-dependently with stretching force in the stretching groups. In contrast, expression of bFGF mRNA showed statistically significant reduction in both stretching groups, with the largest reduction seen in the 9% stretching group (p<0.01). These results suggest that stretching of PDL cells provokes significant increases in expression of factors promoting osteogenic differentiation and HSP70, which protects PDL cells undergoing mechanical stress and contributes to maintenance of PDL homeostasis. However, expression of bFGF was restrained. Reduced expression of bFGF mRNA suggested that there was an optimum magnitude of stretching force for increasing expression.