Takashi Kamakura

BRASSINOSTEROID UPREGULATED1, Encoding a Helix-Loop-Helix Protein, Is a Novel Gene Involved in Brassinosteroid Signaling and Controls Bending of the Lamina Joint in Rice

Brassinosteroids (BRs) are involved in many developmental processes and regulate many subsets of downstream genes throughout the plant kingdom. However, little is known about the BR signal transduction and response network in monocots. To identify novel BR-related genes in rice (Oryza sativa), we monitored the transcriptomic response of the brassinosteroid deficient1 (brd1) mutant, with a defective BR biosynthetic gene, to brassinolide treatment. Here, we describe a novel BR-induced rice gene BRASSINOSTEROID UPREGULATED1 (BU1), encoding a helix-loop-helix protein. Rice plants overexpressing BU1 (BU1:OX) showed enhanced bending of the lamina joint, increased grain size, and resistance to brassinazole, an inhibitor of BR biosynthesis. In contrast to BU1:OX, RNAi plants designed to repress both BU1 and its homologs displayed erect leaves. In addition, compared to the wild type, the induction of BU1 by exogenous brassinolide did not require de novo protein synthesis and it was weaker in a BR receptor mutant OsbriI (Oryza sativa brassinosteroid insensitive1, d61) and a rice G protein alpha subunit (RGA1) mutant d1. These results indicate that BU1 protein is a positive regulator of BR response: it controls bending of the lamina joint in rice and it is a novel primary response gene that participates in two BR signaling pathways through OsBRI1 and RGA1. Furthermore, expression analyses showed that BU1 is expressed in several organs including lamina joint, phloem, and epithelial cells in embryos. These results indicate that BU1 may participate in some other unknown processes modulated by BR in rice.

Blasticidin S-resistance gene (bsr): a novel selectable marker for mammalian cells.

Blasticidin S is a microbial antibiotic that inhibits protein synthesis in both prokaryotes and eukaryotes. The blasticidin S-resistance gene (bsr), isolated from Bacillus cereus K55-S1 strain, was inserted into pSV2 plasmid vector and introduced into cultured mammalian cells by transfection. The bsr gene was integrated into the genome and conferred blasticidin S resistance on HeLa cells. The transfection frequency of the bsr gene was as high as that of the aminoglycoside phosphotransferase gene, the so-called neo gene, which is a representative selectable marker for mammalian cells. Transfectants in which several copies of bsr had been integrated into the genome were highly resistant to blasticidin S. Furthermore, blasticidin S killed the cells more rapidly than G418, which is conventionally used as a selective drug for the neo gene. Thus bsr is concluded to be useful as a drug-resistance marker for mammalian cells.

Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae

Aspergillus terreus produces a unique enzyme, blasticidin S deaminase, which catalyzes the deamination of blasticidin S (BS), and in consequence confers high resistance to the antibiotic. A cDNA clone derived from the structural gene for BS deaminase (BSD) was isolated by transforming Escherichia coli with an Aspergillus cDNA expression library and directly selecting for the ability to grow in the presence of the antibiotic. The complete nucleotide sequene of BSD was determined and proved to contain an open reading frame of 393 bp, encoding a polypeptide of 130 amino acids. Comparison of its nulceotide sequence with that of bsr, the BS deaminase gene isolated from Bacillus cereus, indicated no homology and a large difference in codon usage. The activity of BSD expressed in E. coli was easily quantified by an assay based on spectrophotometric recording. The BSD gene was placed in a shuttle vector for Schizosaccharomyces pombe, downstream of the SV40 early region promoter, and this allowed direct selection with BS at high frequency, following transformation into the yeast. The BSD gene was also employed as a selectable marker for Pyricularia oryzae, which could not be transformed to BS resistance by bsr. These results promise that the BSD gene will be useful as a new dominant selectable marker for eukaryotes.

A Novel Gene, CBP1, Encoding a Putative Extracellular Chitin-Binding Protein, May Play an Important Role in the Hydrophobic Surface Sensing of Magnaporthe grisea During Appressorium Differentiation

The conidial germ tube of the rice blast fungus, Magnaporthe grisea, differentiates a specialized cell, an appressorium, required for penetration into the host plant. Formation of the appressorium is also observed on artificial solid substrata such as polycarbonate. A novel emerging germ tube-specific gene, CBP1 (chitin-binding protein), was found in a cDNA subtractive differential library. CBP1 coded for a putative extracellular protein (signal peptide) with two similar chitin-binding domains at both ends of a central domain with homology to fungal chitin deacetylases and with a C-terminus domain rich in Ser/Thr related extracellular matrix protein such as agglutinin. The consensus sequence of the chitin-binding domain found in CBP1 has never been reported in fungi and is similar to the chitin-binding motif in plant lectins and plant chitinases classes I and IV. CBPI was disrupted in order to identify its function. Null mutants of CBP1 failed to differentiate appressoria normally on artificial surface but succeeded in normally differentiating appressoria on the plant leaf surface. Since the null mutant Cbp1- showed abnormal appressorium differentiation only on artificial surfaces and was sensitive to the chemical inducers, CBP1 seemed to play an important role in the recognition of physical factors on solid surfaces.

Sensitivity of Neurospora crassa to benzimidazoles and N-phenylcarbamates: Effect of amino acid substitutions at position 198 in β-tubulin

Abstract Diethofencarb-resistant revertants have been isolated from the Neurospora crassa mutant F914 which shows both carbendazim (MBC) resistance and diethofencarb sensitivity. Revertant strains FR422 and FR424 were true revertants showing high resistance to N -phenylcarbamates and sensitivity to benzimidazoles, the same as wild-type. However, other revertant strains, FR511, FR513, FR521, FR421, and FRB3, were more resistant to diethofencarb than the strain F914 but less resistant to diethofencarb than the wild-type strain. These moderately resistant strains were also more resistant to benzimidazoles than the parent strain F914, Strains FR421 and FR521 were also resistant to griseofulvin. All the diethofencarb-resistant mutations were linked to the Bml locus that encodes β-tubulin. The β-tubulin gene of FRB3 strain was cloned and sequenced, and an amino acid substitution at the position 198 was found. In the strains FRB3, F914, and wild-type, the amino acids at position 198 were Lys, Gly, and Glu, respectively. A new β-tubulin gene with Ala at position 198 was constructed by site-directed mutagenesis. This gene transformed the wild-type strain to MBC resistance, while transformation of the F914 strain with this gene resulted in growth on the medium containing 0.5 μg/ml diethofencarb but not on that containing 75 μg/ml diethofencarb. The F914 strain transformed by β-tubulin genes of the wild-type strain and the FRB3 strain grew even on medium containing 75 μg/ml diethofencarb. These data suggest that the presence of Glu at position 198 in β-tubulin is responsible for MBC sensitivity. Different amino acid substitutions at position 198 confer different sensitivity to diethofencarb and the order of amino acids which result in increasing sensitivity are Glu, Lys, Ala, and Gly (most sensitive). These approaches should work in clarifying the function of β-tubulin and also aid in the design of new potent fungicides.

Diterpenoid phytoalexin factor, a bHLH transcription factor, plays a central role in the biosynthesis of diterpenoid phytoalexins in rice

Rice (Oryza sativa) produces diterpenoid phytoalexins (DPs), momilactones and phytocassanes as major phytoalexins. Accumulation of DPs is induced in rice by blast fungus infection, copper chloride or UV light. Here, we describe a rice transcription factor named diterpenoid phytoalexin factor (DPF), which is a basic helix-loop-helix (bHLH) transcription factor. The gene encoding DPF is expressed mainly in roots and panicles, and is inducible in leaves by blast infection, copper chloride or UV. Expression of all DP biosynthetic genes and accumulation of momilactones and phytocassanes were remarkably increased and decreased in DPF over-expressing and DPF knockdown rice, respectively. These results clearly demonstrated that DPF positively regulates DP accumulation via transcriptional regulation of DP biosynthetic genes, and plays a central role in the biosynthesis of DPs in rice. Furthermore, DPF activated the promoters of COPALYL DIPHOSPHATE SYNTHASE2 (CPS2) and CYTOCHROME P450 MONOOXYGENASE 99A2 (CYP99A2), whose products are implicated in the biosynthesis of phytocassanes and momilactones, respectively. Mutations in the N-boxes in the CPS2 upstream region, to which several animal bHLH transcription factors bind, decreased CPS2 transcription, indicating that DPF positively regulates CPS2 transcription through the N-boxes. In addition, DPF partly regulates CYP99A2 through the N-box. This study demonstrates that DPF acts as a master transcription factor in DP biosynthesis.

Amino-acid alterations in the beta-tubulin gene of Neurospora crassa that confer resistance to carbendazim and diethofencarb

We have previously shown that increased sensitivity to diethofencarb in the carbendazim(MBC)-resistant F914 strain of Neurospora crassa is caused by a single amino-acid change in β-tubulin, 198Glu to Gly. Three diethofencarb-resistant mutants that are also resistant to MBC were isolated from strain F914. They contained single base-pair-substitution mutations in the β-tubulin gene. The amino acid changes in β-tubulin, Phe from 250Leu, Val from 165Ala, and Ala from 237Thr, were responsible for diethofencarb-resistance in the mutant strains FR511, FR513, and FR421, respectively. The amino acid at position 198 of β-tubulin in these mutants was Gly, which is the same as in strain F914. β-tubulin genes with 198Glu were constructed by site-directed mutagenesis. The altered β-tubulin genes derived from FR511 and FR421 transformed the wild-type strain to resistance to MBC, indicating that 250Phe and 237Ala in β-tubulin are responsible for resistance not only to diethofencarb but also to MBC.

MgLig4, a homolog of Neurospora crassa Mus-53 (DNA ligase IV), is involved in, but not essential for, non-homologous end-joining events in Magnaporthe grisea

In many eukaryotic organisms, the non-homologous end-joining (NHEJ) system is a major pathway for the repair of DNA double-strand breaks (DSBs). DNA ligase IV is a component of the NHEJ system and is strictly required for the NHEJ system in Saccharomyces cerevisiae and in Neurospora crassa. To investigate the functions of DNA Ligase IV in Magnaporthe grisea, we generated deletion mutants of MGLIG4, which encodes a homolog of N. crassa DNA Ligase IV. Mutants (mglig4) showed no defects in asexual or sexual growth, and were fully pathogenic. Compared to the wild-type, mglig4 exhibited weak sensitivity to a DNA-damaging agent, camptothecin. In addition, the frequency of targeted-gene replacement was relatively elevated in mglig4, although this varied in a gene-dependent manner. Surprisingly, non-homologous integration of DNA was frequently observed in mglig4 transformants. Our results demonstrate that MgLig4 is involved in, but not essential for, the NHEJ system in M. grisea.

Expression of the blasticidin S deaminase gene (bsr) in tobacco: fungicide tolerance and a new selective marker for transgenic plants.

SummaryBlasticidin S (BS), a fungicide of microbial origin, is used for the practical control of rice blast disease. It has broad antimicrobial activity but occasionally exhibits adverse phytotoxic effects on some dicot plants. An inactivating enzyme, BS deaminase, was discovered in the BS resistant strain, Bacillus cereus K55-S1, and the structural gene, bsr, for the enzyme has been cloned. We introduced the bsr gene into tobacco plants using the Ti plasmid vector system and demonstrated that the bsr gene conferred a BS resistant phenotype to the plants. Thus the bsr gene could be useful as a selective marker for plant transformation and provides an example for a new approach to the solution of phytotoxicity problems associated with the use of some types of fungicide.

TGF-β3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells

Transforming growth factor-βs (TGF-βs), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-β signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-β signaling components in taste epithelia and found that the TGF-β3 mRNA was specifically expressed in taste buds. Type II TGF-βs receptor (TβR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-β3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-β3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-β3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription–polymerase chain reaction assay, we found that the TGF-β3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-β3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin–Cdk complexes. Taken together, these results suggested that the TGF-β3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.