(T.A.) 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.Brassinosteroids (BRs) are essential growth regulators, involved in many physiological processes, e.g. cell expansion and division, vascular bundle differentiation, skotomorphogenesis, flowering, senescence, abiotic, and biotic stresses (Szekeres et al

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.

SUMMARYRice (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 DIPHO-SPHATE SYNTHASE2 (CPS2) and CYTOCHROME P450 MONOOXYGENASE 99A2 (CYP99A2), whose products are implicated in the biosynthesis of phytocassanes and momilactones, respectively. Mutations in the Nboxes 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.

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.

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 sequence 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 nucleotide 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 result promise that the BSD gene will be useful as a new dominant selectable marker for eukaryotes.

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The rice blast fungus Magnaporthe oryzae differentiates a specialized infection structure called an appressorium to invade rice cells. In this report, we show that CBP1, which encodes a chitin-deacetylase, is involved in the induction phase of appressorium differentiation. We demonstrate that the enzymatic activity of Cbp1 is critical for appressorium formation. M. oryzae has six CDA homologues in addition to Cbp1, but none of these are indispensable for appressorium formation. We observed chitosan localization at the fungal cell wall using OGA488. This observation suggests that Cbp1-catalysed conversion of chitin into chitosan occurs at the cell wall of germ tubes during appressorium differentiation by M. oryzae. Taken together, our results provide evidence that the chitin deacetylase activity of Cbp1 is necessary for appressorium formation.