Publications by authors named "Hidetaka Kaya"

The depletion of cellular zinc (Zn) adversely affects plant growth. Plants have adaptation mechanisms for Zn-deficient conditions, inhibiting growth through the action of transcription factors and metal transporters. We previously identified three defensin-like (DEFL) proteins (DEFL203, DEFL206 and DEFL208) that were induced in Arabidopsis thaliana roots under Zn-depleted conditions.

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Reactive oxygen species (ROS) produced by plant NADPH oxidases, respiratory burst oxidase homologs (RBOHs), play key roles in biotic and abiotic stress responses and development in plants. While properly controlled amounts of ROS function as signaling molecules, excessive accumulation of ROS can cause undesirable side effects due to their ability to oxidize DNA, lipids, and proteins. To limit the damaging consequences of unrestricted ROS accumulation, RBOH activity is tightly controlled by post-translational modifications (PTMs) and protein-protein interactions.

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To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the () gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient mediated gene transfer. A gene was introduced into and together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of homologs (s). All three sgRNAs directed the introduction of InDels into s, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency.

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The CRISPR/Cas9 system is now commonly employed for genome editing in various plants such as Arabidopsis, rice and tobacco. In general, in genome editing of the Arabidopsis genome, the SpCas9 and guide RNA genes are introduced into the genome by the floral dip method. Mutations induced in the target sequence by SpCas9 are confirmed after selecting transformants by screening the T1 seed population.

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In the present study, we have shown the transcriptional changes in a chlorosis model transgenic tobacco plant, i-amiCHLI, in which an artificial micro RNA is expressed in a chemically inducible manner to silence the expression of genes encoding a subunit of a chlorophyll biosynthetic enzyme. Comparison to the inducer-treated and untreated control non-transformants and untreated i-amiCHLI revealed that 3568 and 3582 genes were up- and down-regulated, respectively, in the inducer-treated i-amiCHLI plants. Gene Ontology enrichment analysis of these differentially expressed genes indicated the upregulation of the genes related to innate immune responses, and cell death pathways, and the downregulation of genes for photosynthesis, plastid organization, and primary and secondary metabolic pathways in the inducer-treated i-amiCHLI plants.

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RNA-seq analysis of a transgenic tobacco plant, i-hpHSP90C, in which chloroplast HSP90C genes can be silenced in an artificially inducible manner resulting in the development of chlorosis, revealed the up- and downregulation of 2746 and 3490 genes, respectively. Gene ontology analysis of these differentially expressed genes indicated the upregulation of ROS-responsive genes; the activation of the innate immunity and cell death pathways; and the downregulation of genes involved in photosynthesis, plastid organization, and cell cycle. Cell death was confirmed by trypan blue staining and electrolyte leakage assay, and the HO production was confirmed by diaminobenzidine staining.

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FLOWERING LOCUS T (FT) is an essential component of florigen in Transcription of is induced in leaves, and the resulting FT protein is transported to the shoot apex, in which it initiates floral development. Previous analyses suggest that, together with the b-ZIP transcription factor FD, FT regulates the transcription of downstream targets such as () in floral anlagen. However, conclusive evidence that FT is transported to the shoot apex to form an FT-FD complex is lacking.

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Reactive oxygen species (ROS) produced by NADPH oxidases, called respiratory burst oxidase homologs (Rbohs), play crucial roles in development as well as biotic and abiotic stress responses in plants. Arabidopsis has 10 Rboh genes, AtRbohA to AtRbohJ. Five AtRbohs (AtRbohC, -D, -F, -H and -J) are synergistically activated by Ca -binding and protein phosphorylation to produce ROS that play various roles in planta, although the activities of the other Rbohs remain unknown.

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Streptococcus pyogenes Cas9 (SpCas9) is widely used for genome editing and requires NGG as a protospacer adjacent motif (PAM). Here, we show that the engineered SpCas9 (SpCas9-NGv1) can efficiently mutagenize endogenous target sites with NG PAMs in the rice and Arabidopsis genomes. Furthermore, we demonstrate that the SpCas9-NGv1 nickase fused to cytidine deaminase mediates C-to-T substitutions near the 5' end of the target sequence.

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Split-protein methods-where a protein is split into two inactive fragments that must re-assemble to form an active protein-can be used to regulate the activity of a given protein and reduce the size of gene transcription units. Here, we show that a Staphylococcus aureus Cas9 (SaCas9) can be split, and that split-SaCas9 expressed from Agrobacterium can induce targeted mutagenesis in Nicotiana benthamiana. Since SaCas9 is smaller than the more commonly used Cas9 derived from Streptococcus pyogenes, the split-SaCas9 provides the smallest tool yet for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) plant genome editing.

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Genome editing in plants becomes popular since the advent of sequence-specific nucleases (SSNs) that are simple to set up and efficient in various plant species. Although transcription activator-like effector nucleases (TALENs) are one of the most prevalent SSNs and have a potential to provide higher target specificity by their dimeric property, TALENs are sensitive to methylated cytosines that are present not only in transposons but also in active genes in plants. In mammalian cells, the methylation sensitivity of TALENs could be overcome by using a base-recognition module (N) that has a higher affinity to methylated cytosine.

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CRISPR/Cas9 systems are nowadays applied extensively to effect genome editing in various organisms including plants. CRISPR from Prevotella and Francisella 1 (Cpf1) is a newly characterized RNA-guided endonuclease that has two distinct features as compared to Cas9. First, Cpf1 utilizes a thymidine-rich protospacer adjacent motif (PAM) while Cas9 prefers a guanidine-rich PAM.

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The CRISPR/Cas9 system is an efficient and convenient tool for genome editing in plants. Cas9 nuclease derived from Streptococcus pyogenes (Sp) is commonly used in this system. Recently, Staphylococcus aureus Cas9 (SaCas9)-mediated genome editing was reported in human cells and Arabidopsis.

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In many flowering plants, the transition to flowering is primarily affected by seasonal changes in day length (photoperiod). An inductive photoperiod promotes flowering via synthesis of a floral stimulus, called florigen. In Arabidopsis thaliana, the FLOWERING LOCUS T (FT) protein is an essential component of florigen, which is synthesized in leaf phloem companion cells and is transported through phloem tissue to the shoot apical meristem where floral morphogenesis is initiated.

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Reactive oxygen species (ROS) accumulate at the tip of growing pollen tubes. In Arabidopsis, NADPH oxidases RbohH and RbohJ are localized at the plasma membrane of pollen tube tip and produce ROS in a Ca(2+)-dependent manner. The ROS produced by Rbohs and Ca(2+) presumably play a critical role in the positive feedback regulation that maintains the tip growth.

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In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana.

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Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in plant environmental responses. Arabidopsis thaliana NADPH oxidase AtRbohF-mediated ROS-production is involved in abiotic stress responses. Because overproduction of ROS is highly toxic to cells, the activity of AtRbohF needs to be tightly regulated in response to diverse stimuli.

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The plant NADPH oxidases, known as respiratory burst oxidase homologues (Rbohs), play an indispensable role in a wide array of cellular and developmental processes. Arabidopsis thaliana RbohF (AtRbohF)-mediated production of reactive oxygen species (ROS) is involved in biotic and abiotic stress responses. Because of the toxicity of excess amount of ROS, the ROS-producing activity of Rbohs is speculated to be negatively regulated.

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Reactive oxygen species (ROS) produced by plant NADPH oxidases (NOXes) are important in plant innate immunity. The Oryza sativa respiratory burst oxidase homologue B (OsRbohB) gene encodes a NOX the regulatory mechanisms of which are largely unknown. Here, we used a heterologous expression system to demonstrate that OsRbohB shows ROS-producing activity.

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Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in signalling and development. Given the high toxicity of ROS, their production is tightly regulated. In Arabidopsis, respiratory burst oxidase homologue F (AtrbohF) encodes NADPH oxidase.

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The specification and maintenance of growth sites are tightly regulated during cell morphogenesis in all organisms. ROOT HAIR DEFECTIVE 2 reduced nicotinamide adenine dinucleotide phosphate (RHD2 NADPH) oxidase-derived reactive oxygen species (ROS) stimulate a Ca2+ influx into the cytoplasm that is required for root hair growth in Arabidopsis thaliana. We found that Ca2+, in turn, activated the RHD2 NADPH oxidase to produce ROS at the growing point in the root hair.

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Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91(phox)) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91(phox)/NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca(2+). However, the significance of Ca(2+) binding to the EF-hand motifs on ROS production has been unclear.

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Chromatin assembly factor 1 (CAF-1) is involved in nucleo some assembly following DNA replication and nucleotide excision repair. In Arabidopsis thaliana, the three CAF-1 subunits are encoded by FAS1, FAS2 and, most likely, MSI1, respectively. In this study, we asked whether genomic stability is altered in fas1 and fas2 mutants that are lacking CAF-1 activity.

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