Takayuki Asano

A rice calcium‐dependent protein kinase OsCPK12 oppositely modulates salt‐stress tolerance and blast disease resistance

Calcium-dependent protein kinases (CDPKs) regulate the downstream components in calcium signaling pathways. We investigated the effects of overexpression and disruption of an Oryza sativa (rice) CDPK (OsCPK12) on the plants response to abiotic and biotic stresses. OsCPK12-overexpressing (OsCPK12-OX) plants exhibited increased tolerance to salt stress. The accumulation of hydrogen peroxide (H(2) O(2) ) in the leaves was less in OsCPK12-OX plants than in wild-type (WT) plants. Genes encoding reactive oxygen species (ROS) scavenging enzymes (OsAPx2 and OsAPx8) were more highly expressed in OsCPK12-OX plants than in WT plants, whereas the expression of the NADPH oxidase gene, OsrbohI, was decreased in OsCPK12-OX plants compared with WT plants. Conversely, a retrotransposon (Tos17) insertion mutant, oscpk12, and plants transformed with an OsCPK12 RNA interference (RNAi) construct were more sensitive to high salinity than were WT plants. The level of H(2) O(2) accumulation was greater in oscpk12 and OsCPK12 RNAi plants than in the WT. These results suggest that OsCPK12 promotes tolerance to salt stress by reducing the accumulation of ROS. We also observed that OsCPK12-OX seedlings had increased sensitivity to abscisic acid (ABA) and increased susceptibility to blast fungus, probably resulting from the repression of ROS production and/or the involvement of OsCPK12 in the ABA signaling pathway. Collectively, our results suggest that OsCPK12 functions in multiple signaling pathways, positively regulating salt tolerance and negatively modulating blast resistance.

Comparative analysis of root transcriptome profiles of two pairs of drought-tolerant and susceptible rice near-isogenic lines under different drought stress

BackgroundPlant roots are important organs to uptake soil water and nutrients, perceiving and transducing of soil water deficit signals to shoot. The current knowledge of drought stress transcriptomes in rice are mostly relying on comparative studies of diverse genetic background under drought. A more reliable approach is to use near-isogenic lines (NILs) with a common genetic background but contrasting levels of resistance to drought stress under initial exposure to water deficit. Here, we examined two pairs of NILs in IR64 background with contrasting drought tolerance. We obtained gene expression profile in roots of rice NILs under different levels of drought stress help to identify genes and mechanisms involved in drought stress.ResultsGlobal gene expression analysis showed that about 55% of genes differentially expressed in roots of rice in response to drought stress treatments. The number of differentially expressed genes (DEGs) increased in NILs as the level of water deficits, increased from mild to severe condition, suggesting that more genes were affected by increasing drought stress. Gene onthology (GO) test and biological pathway analysis indicated that activated genes in the drought tolerant NILs IR77298-14-1-2-B-10 and IR77298-5-6-B-18 were mostly involved in secondary metabolism, amino acid metabolism, response to stimulus, defence response, transcription and signal transduction, and down-regulated genes were involved in photosynthesis and cell wall growth. We also observed gibberellic acid (GA) and auxin crosstalk modulating lateral root formation in the tolerant NILs.ConclusionsTranscriptome analysis on two pairs of NILs with a common genetic background (~97%) showed distinctive differences in gene expression profiles and could be effective to unravel genes involved in drought tolerance. In comparison with the moderately tolerant NIL IR77298-5-6-B-18 and other susceptible NILs, the tolerant NIL IR77298-14-1-2-B-10 showed a greater number of DEGs for cell growth, hormone biosynthesis, cellular transports, amino acid metabolism, signalling, transcription factors and carbohydrate metabolism in response to drought stress treatments. Thus, different mechanisms are achieving tolerance in the two tolerant lines.

Functional characterisation of OsCPK21, a calcium-dependent protein kinase that confers salt tolerance in rice

Calcium acts as a messenger in various signal transduction pathways in plants. Calcium-dependent protein kinases (CDPKs) play important roles in regulating downstream components in calcium signaling pathways. In rice, the CDPKs constitute a large multigene family consisting of 29 genes, but the biological functions and functional divergence or redundancy of most of these genes remain unclear. Using a mini-scale full-length cDNA overexpressor (FOX) gene hunting system, we generated 250 independent transgenic rice plants overexpressing individual rice CDPKs (CDPK FOX-rice lines). These CDPK FOX-rice lines were screened for salt stress tolerance. The survival rate of the OsCPK21-FOX plants was higher than that of wild-type (WT) plants grown under high salinity conditions. The inhibition of seedling growth by abscisic acid (ABA) treatment was greater in the OsCPK21-FOX plants than in WT plants. Several ABA- and high salinity-inducible genes were more highly expressed in the OsCPK21-FOX plants than in WT plants. These results suggest that OsCPK21 is involved in the positive regulation of the signaling pathways that are involved in the response to ABA and salt stress.

CDPK-mediated abiotic stress signaling

Calcium-dependent protein kinases (CDPKs) constitute a large multigene family in various plant species. CDPKs have been shown to have important roles in various physiological processes, including plant growth and development and abiotic and biotic stress responses in plants. Functional analysis using gain-of-function and loss-of-function mutants has revealed the biological function of CDPKs in planta. Several CDPKs have been shown to be essential factors in abiotic stress tolerance, positively or negatively regulating stress tolerance by modulating ABA signaling and reducing the accumulation of reactive oxygen species (ROS). This review summarizes recent results describing the biological function of CDPKs that are involved in abiotic stress tolerance.

Engraftment and tumor formation after allogeneic in utero transplantation of primate embryonic stem cells

Background. To achieve human embryonic stem (ES) cell-based transplantation therapies, allogeneic transplantation models of nonhuman primates would be useful. We have prepared cynomolgus ES cells genetically marked with the green fluorescent protein (GFP). The cells were transplanted into the allogeneic fetus, taking advantage of the fact that the fetus is so immunologically immature as not to induce immune responses to transplanted cells and that fetal tissue compartments are rapidly expanding and thus providing space for the engraftment. Methods. Cynomolgus ES cells were genetically modified to express the GFP gene using a simian immunodeficiency viral vector or electroporation. These cells were transplanted in utero with ultrasound guidance into the cynomolgus fetus in the abdominal cavity (n=2) or liver (n=2) at the end of the first trimester. Three fetuses were delivered 1 month after transplantation, and the other, 3 months after transplantation. Fetal tissues were examined for transplanted cell progeny by quantitative polymerase chain reaction and in situ polymerase chain reaction of the GFP sequence. Results. A fluorescent tumor, obviously derived from transplanted ES cells, was found in the thoracic cavity at 3 months after transplantation in one fetus. However, transplanted cell progeny were also detected (∼1%) without teratomas in multiple fetal tissues. The cells were solitary and indistinguishable from surrounding host cells. Conclusions. Transplanted cynomolgus ES cells can be engrafted in allogeneic fetuses. The cells will, however, form a tumor if they “leak” into an improper space such as the thoracic cavity.

In vivo selective expansion of gene-modified hematopoietic cells in a nonhuman primate model

A major problem limiting hematopoietic stem cell (HSC) gene therapy is the low efficiency of gene transfer into human HSCs using retroviral vectors. Strategies, which would allow in vivo expansion of gene-modified hematopoietic cells, could circumvent the problem. To this end, we developed a selective amplifier gene (SAG) consisting of a chimeric gene composed of the granulocyte colony-stimulating factor (G-CSF) receptor gene and the estrogen receptor gene hormone-binding domain. We have previously demonstrated that primary bone marrow progenitor cells transduced with the SAG could be expanded in response to estrogen in vitro. In the present study, we evaluated the efficacy of the SAG in the setting of a clinically applicable cynomolgus monkey transplantation protocol. Cynomolgus bone marrow CD34+ cells were transduced with retroviral vectors encoding the SAG and reinfused into each myeloablated monkey. Three of the six monkeys that received SAG transduced HSCs showed an increase in the levels of circulating progeny containing the provirus in vivo following administration of estrogen or tamoxifen without any serious adverse effects. In one monkey examined in detail, transduced hematopoietic progenitor cells were increased by several-fold (from 5% to 30%). Retroviral integration site analysis revealed that this observed increase was polyclonal and no outgrowth of a dominant single clonal population was observed. These results demonstrate that the inclusion of our SAG in the retroviral construct allows selective in vivo expansion of genetically modified cells by a non-toxic hormone treatment.

A new flap design for tongue reconstruction after total or subtotal glossectomy in thin patients

OBJECTIVE For tongue reconstruction after total or subtotal glossectomy, a rectus abdominis musculocutaneous flap is often used to obtain sufficient flap volume. However, thin patients often have too little fat tissue to ensure adequate flap volume. For this reason we developed a new flap design to compensate for insufficient flap volume in thin patients. METHODS In this series, total or subtotal glossectomy was performed in 20 thin men with a mean age of 58.3 years. The patients had a mean body mass index of 18.22 kg/m(2) and most were considered emaciated. The defects were reconstructed using a rectus abdominis musculocutaneous flap with two skin islands. The first skin island was used to reconstruct the mucosal defect, and the second was de-epithelialised and used to increase flap volume. RESULTS Flaps were transferred successfully in 19 out of 20 patients. Most patients could tolerate more than a soft diet without severe aspiration and could engage in conversation. However, four patients required total laryngectomy or a permanent stoma owing to severe aspiration. In this series, the larynx could be preserved in 80% of thin patients, and satisfactory postoperative oral function was obtained. CONCLUSION The most important point for obtaining satisfactory oral function is to reconstruct a tongue with a protuberant shape and sufficient volume. We could maintain sufficient flap volume with the de-epithelialised skin island of a rectus abdominis musculocutaneous free flap. We believe our new flap design is effective for tongue reconstruction in thin patients.

Introduction of the green fluorescent protein gene into hematopoietic stem cells results in prolonged discrepancy of in vivo transduction levels between bone marrow progenitors and peripheral blood cells in nonhuman primates.

The green fluorescent protein (GFP) has proven a useful marker in retroviral gene transfer studies targeting hematopoietic stem cells (HSCs) in mice. However, several investigators have reported very low in vivo peripheral blood marking levels in nonhuman primates after transplantation of HSCs transduced with the GFP gene. We retrovirally marked cynomolgus monkey HSCs with the GFP gene, and tracked in vivo marking levels within both bone marrow progenitor cells and mature peripheral blood cells following autologous transplantation after myeloablative conditioning.

Molecular characterization of ONAC300, a novel NAC gene specifically expressed at early stages in various developing tissues of rice

Members of the plant-specific gene family referred to as the NAC family (for NAM-ATAF-CUC-related) are involved in various functions including the regulation of plant development. However, no detailed molecular characterization of any member of the NAC family has yet been reported from monocots. Here, we report such a characterization of ONAC300, a novel NAC-family gene identified using a cDNA cloned from microdissected phloem cells of rice. The predicted ONAC300 protein sequence falls into the NAM subgroup, which also contains the proteins CUC1 and CUC2 from Arabidopsis, CUP from snapdragon, CmNACP from pumpkin and NAM from petunia. High levels of ONAC300 mRNA were detected by in situ hybridization in developing shoot apical meristem (SAM) and in the associated young leaves. The use of an ONAC300:: GUS reporter gene revealed that the ONAC300 promoter was expressed predominantly in developing vascular tissues of the leaves and roots. The construct was also expressed in anther filaments, rachis and carpel styles. RT-PCR analysis further revealed that the levels of ONAC300 transcripts were higher in leaves, roots and culms than in panicles. The observed expression pattern of ONAC300 is quite different from those of the dicot NAC genes previously reported. Thus, ONAC300 is a novel member of the NAC family which is expressed at very early developmental stages in the shoot, root and flower, as well as in the mature phloem of vascular tissues in rice.

Genetic Manipulation of Primate Embryonic and Hematopoietic Stem Cells with Simian Lentivirus Vectors

During the past several years, many articles have described how human embryonic stem (ES) cells and adult hematopoietic stem cells (HSCs) can differentiate into cardiac muscle, blood vessels, and various other types of cells. The articles raised the expectation that these stem cells may become useful for the treatment of a variety of diseases, including cardiovascular diseases. Genetic manipulation of ES cells and HSCs would be important for such future applications of the cells. Until now, retroviral vectors have been used primarily for stable expression of transgenes in murine ES cells and HSCs. Because murine models may not predict reliably the biology of ES cells and HSCs in humans, we have utilized primate ES cells and HSCs as targets of gene transfer. We have shown that primate ES cells and HSCs can be transduced efficiently with lentiviral vectors derived from the simian immunodeficiency virus, and that the high transgene expression persists without transcriptional silencing. This highly efficient gene transfer method allows for safe and faithful gene delivery to primate ES cells and HSCs to test potential research and therapeutic applications.